Apoptosis In Blast Cells Research Articles

Abstract 3659: The AKT inhibitor MK-2206 promotes apoptosis in acute myeloid leukemia cells

Abstract The need for improved therapies for acute myeloid leukemia (AML) is urgent as the disease is still highly fatal. Protein Kinase B (AKT) activation has been shown to predict poor clinical outcome for patients with AML. In 253 newly diagnosed AML cases, we have found that AKT phosphorylation on Thr308 analyzed by reverse-phase protein arrays was significantly higher in patients with unfavorable cytogenetics (p=0.037). The adverse prognostic factor of high levels of pAKT was observed among patients with intermediate-risk cytogenetics (p=0.013), as it was associated with an inferior survival (median survival 44.2 weeks) compared to patients with low or intermediate pAKT (78.7 weeks). Recently, Merck Sharp & Dohme Corp. has developed a compound, MK-2206, that is a highly specific allosteric inhibitor for all three AKT isoforms. In the current study, we have determined that MK-2206 as a single agent used at clinically achievable concentrations suppresses cell growth of a variety of AML derived cell lines including OCI-AML3, U937, and MOLM13. MK-2206 can also induce apoptosis in blast cells derived from AML patients. Consistent with its role as an AKT inhibitor, the drug suppresses phosphorylation of AKT at serine 473 and inhibits phosphorylation of AKT downstream targets including S6 ribosomal protein, 4-EBP1, and PRAS40 in the AML cells. We recently identified that low expression of the PP2A isoform that acts as the AKT phosphatase (i.e. the one containing the B55α subunit) in AML negatively correlates with AKT T308 phosphorylation and activation in a cohort of 511 AML patients (Ruvolo et al Leukemia, 2011). Patients with low B55α expression exhibited shorter complete remission duration. We suppressed B55α expression by shRNA in OCI-AML3 cells to determine if loss of the AKT phosphatase promotes AKT activation and chemoresistance. Cells with reduced B55α exhibited higher levels of AKT phosphorylation and were more resistant to killing by the conventional chemotherapy agent AraC. OCI-AML3 B55α shRNA transductant cells were also more resistant to MK-2206 compared to cells expressing control non-specific shRNA. These results suggest that AKT phosphatase activity in AML may affect response to MK-2206. A recent study in liver cancer demonstrated that MK-2206 synergized with the multi-kinase inhibitor Sorafenib. Since Sorafenib targets the Fms-like tyrosine kinase 3 (FLT3) and mutations that activate the kinase in AML confer poor prognosis, we examined if MK-2206 could synergize with Sorafenib to kill leukemia cells with mutation of FLT3 internal tandem duplication (ITD) mutation. The combination of MK-2206 and Sorafenib was effective at killing MOLM13 cells and AML blast cells with the FLT3-ITD. Taken together, these findings suggest that MK-2206 may be effective to block AKT signaling pathways and could be beneficial as a therapeutic agent perhaps as a single agent or in combination with a drug such as Sorafenib in the therapy of AML. 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 3659. doi:1538-7445.AM2012-3659

EBV-associated post-transplantation B-cell lymphoproliferative disorder following allogenic stem cell transplantation for acute lymphoblastic leukaemia: tumor regression after reduction of immunosuppression - a case report

Epstein-Barr virus (EBV)-associated B-cell post-transplantation lymphoproliferative disorder (PTLD) is a severe complication following stem cell transplantation. This is believed to occur as a result of iatrogenic immunosuppression leading to a relaxation of T-cell control of EBV infection and thus allowing viral reactivation and proliferation of EBV-infected B-lymphocytes. In support of this notion, reduction of immunosuppressive therapy may lead to regression of PTLD.We present a case of an 18-year-old male developing a monomorphic B-cell PTLD 2 months after receiving an allogenic stem cell transplant for acute lymphoblastic leukemia. Reduction of immunosuppressive therapy led to regression of lymphadenopathy. Nevertheless, the patient died 3 months afterwards due to extensive graft-vs.-host-disease and sepsis. As a diagnostic lymph node biopsy was performed only after reduction of immunosuppressive therapy, we are able to study the histopathological changes characterizing PTLD regression. We observed extensive apoptosis of blast cells, accompanied by an abundant infiltrate comprising predominantly CD8-positive, Granzyme B-positive T-cells. This observation supports the idea that regression of PTLD is mediated by cytotoxic T-cells and is in keeping with the observation that T-cell depletion, represents a major risk factor for the development of PTLD.

Role of the PI3K/AKT and mTOR signaling pathways in acute myeloid leukemia

The PI3K/AKT and mTOR signaling pathways are activated in acute myeloid leukemia, including in the more immature leukemic populations. Constitutive PI3K activation is detectable in 50% of acute myeloid leukemia samples whereas mTORC1 is activated in all cases of this disease. In leukemic cells, the PI3K activity relates to the expression of the p110delta isoform of class IA PI3K. Constitutive PI3K activation is the result of autocrine IGF-1/IGF-1R signaling in 70% of acute myeloid leukemia samples but specific inhibition of this pathway does not induce apoptosis. Specific inhibition of PI3K/AKT or mTORC1 alone in vitro has anti-leukemic effects which are essentially exerted via the suppression of proliferation. However, as mTORC1 activation is independent of PI3K/AKT in acute myeloid leukemia, dual PI3K and mTOR inhibitors may induce apoptosis in blast cells. Moreover, mTORC1 inhibition using sirolimus overactivates PI3K/AKT via the upregulation of IRS2 expression and by favoring IGF-1/IGF-1R autocrine signaling. Recent data also indicate that mTORC1 does not control protein translation in acute myeloid leukemia. These results open the way for the design of direct inhibitors of protein synthesis as novel acute myeloid leukemia therapies and also for the development of second generation mTOR inhibitors (the TORKinhibs).

PI3K Signaling Pathway Activation Predicts Class I PI3K Inhibitor GDC-0941 Sensitivity in AML.

Abstract 1057Poster Board I-79The PI3K-Akt signal transduction pathway plays a key role in the pathogenesis of many human cancers. In AML malignancy, deregulation of upstream receptor tyrosine kinases such as FLT3, c-Kit, and c-FMS or mutations in K-Ras, N-Ras, B-Raf and CEBPA genes lead to activation of PI3K-Akt signaling to promote cell survival and cell growth. A highly selective Class I PI3K inhibitor, GDC-0941, provides exciting therapeutic opportunities for targeting this pathway in AML. Here we show that GDC-0941 significantly inhibits the viability of the majority of a large panel of AML cell lines tested in vitro (80% or 19/24) at a concentration of < 1 uM. Because not all AML cell lines responded to GDC-0941, we show that PI3K-Akt pathway activation, evidenced by basal pAkt level, can serve as a potential predictive biomarker for GDC-0941 in AML in that sensitive cell lines displayed higher level of pAkt relative to resistant cell lines. Consistently, GDC-0941 treatment leads to decreased pAkt, and therefore the down-regulation of this important pro-survival signaling. Our further analysis shows that GDC-0941 treatment can induce apoptosis and/or cell cycle arrest. We also obtained fresh AML tumor samples to test whether GDC-0941 can similarly induces apoptosis in blast cells and showed that GDC-0941 treatment results in a down-regulation of pAkt level and increased apoptosis. Other PD biomarkers such as phospho-BAD level and Bim expression are both consistent with the observed apoptotic responses. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) inhibitor, rapamycin, synergizes with GDC-0941 to produce an increased amount of apoptosis in several AML cell lines tested. This is likely due to the fact that long-term treatment with rapamycin induces the sensitivity of the PI3K –Akt signaling pathway by releasing the negative feedback loop of mTORC1-S6K-IRS1/2 module. Importantly, in some AML cell lines we observe synergies between GDC-0941 and AraC. Interestingly, while AraC alone does not induce apoptosis in AML cell lines with PTEN loss or mutation, the synergy between AraC and GDC-0941 comes from increased apoptotic response, suggesting that GDC-0941 can synergize with chemo agents that induces S/G2 cell cycle arrest. Together, our preclinical data suggest that GDC-0941 may be used as a targeted therapy in AML patients as a single agent or in combination with other chemotherapies in clinic. Disclosures:Ma:Genentech Inc.: Employment. Du:Genentech, Inc.: Employment, Equity Ownership. Sun:Genentech Inc.: Employment. Shi:Genentech, Inc.: Employment, Equity Ownership. Friedman:Genentech Inc.: Employment. Dornan:Genentech, Inc.: Employment, Equity Ownership. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

Improved Selectivity against Acute Myeloid Leukemia (AML) Blasts Over Normal Hematopoietic Progenitors for Cytarabine: Daunorubicin Delivered as CPX-351 Liposome Injection.

Abstract 2071Poster Board II-48 Background:Combinations of cytarabine (Ara-C) and daunorubicin (DNR) have been the mainstay of induction chemotherapy for AML for three decades. Although many patients achieve a complete remission (CR), at least 20% have leukemia that is refractory (NR) to this treatment and the majority of the remainder eventually relapse with chemotherapy resistant disease. One of the main mechanisms of multidrug resistance in AML is over-expression of ATP-binding cassette (ABC) transporters such as MDR-1 and BCRP-1. CPX-351 is a liposomal formulation of Ara-C and DNR in which the ratio of the two drugs encapsulated (5:1, mol:mol) has been designed to maximize synergistic interactions. In addition, preclinical studies demonstrated a prolonged half-life of 24 to 36h and markedly improved in vivo efficacy against animal leukemia models for CPX-351. More recently, encouraging Phase 1 trial results in patients with advanced acute leukemia have resulted in continued clinical development of this drug in a Phase 2 setting. The purpose of this study was to compare the relative sensitivity of colony forming cells (CFC) from AML patient blast samples and normal blood (PB) and marrow (BM) to CPX-351 and the same concentrations of free Ara-C and DNR (FD). Investigations of the relative sensitivity of AML blasts from CR and NR patients to CPX-351 as well as the intracellular accumulation and sub-cellular localization of the liposomal drugs were undertaken in an attempt to elucidate the basis for the putative improved efficacy observed with CPX-351. Methods:A panel of AML patient blast samples (including CR and NR to induction chemotherapy) as well as normal PB and BM samples were assessed for drug sensitivity by incubating cells for 24h in various concentrations of CPX-351 or the same concentrations of FD prior to plating in CFC assays with or without ABC transporter inhibition by cyclosporine A (CA). Quantitative RT-PCR was used to measure expression of the ABC transporter proteins MDR-1 and BCRP-1. HPLC and confocal microscopy were used to study the cellular drug uptake and subcellular distribution of CPX-351, respectively. Results:Comparison of the IC50 values obtained against AML-CFC for CPX-351 and FD demonstrated no significant difference in progenitor kill despite the fact that drug release from CPX-351 liposomes in media was negligible under exposure conditions, suggesting direct uptake of CPX-351 liposomes by the leukemia cells. However, CPX-351 was approximately 3-fold less cytotoxic against normal PB and BM CFC than FD. CPX-351 was also 3-9-fold more potent against AML-CFC than against normal CFC while the same comparison for FD showed less than a 3-fold difference. HPLC demonstrated accumulation of CPX-351 in AML blasts and confocal microscopy showed the presence of liposomes in the cytoplasm and daunorubicin within the cytoplasm and nucleus of these cells. AML blasts from NR patients showed expression of MDR-1 and/or BCRP-1 which was 3.5 to >200-fold higher than that shown in blasts from CR patients. There was no consistent difference in qualitative induction of AML blast cell apoptosis between CPX-351 and FD with either the CR or the NR samples. However, the addition of CA, which had no effect on the CR sample treated with either CPX-351 or FD, rendered the NR samples more susceptible to both CPX-351 and FD. Interestingly, the CA-induced increase in cell kill was greater with CPX-351 than with FD. Conclusions:CPX-351 exhibits less toxicity against normal PB and BM progenitors than against AML blast CFC and this difference is greater than that seen with conventional cytarabine and daunorubicin. The CPX-351 liposomes appear to enter blast cells and subsequently release their contents where they can be detected in both the cytoplasm and the nucleus. These data suggest an enhanced specificity for the CPX-351 liposomal formulation of cytarabine and daunorubicin against AML over normal hematopoietic progenitors as well as potential differences in cellular uptake and metabolism as compared to the free chemotherapy drugs that may provide a therapeutic advantage. Disclosures:No relevant conflicts of interest to declare.

Protein synthesis is resistant to rapamycin and constitutes a promising therapeutic target in acute myeloid leukemia

AbstractThe deregulation of translation markedly contributes to the malignant phenotype in cancers, and the assembly of the translation initiating complex eIF4F is the limiting step of this process. The mammalian Target of Rapamycin Complex 1 (mTORC1) is thought to positively regulate eIF4F assembly and subsequent oncogenic protein synthesis through 4E-BP1 phosphorylation. We showed here that the translation inhibitor 4EGI-1 decreased the clonogenic growth of leukemic progenitors and induced apoptosis of blast cells, with limited toxicity against normal hematopoiesis, which emphasize the importance of translation deregulation in acute myeloid leukemia (AML) biology. However, the mTORC1 inhibitor RAD001 (a rapamycin derivate) did not induce AML blast cell apoptosis. We herein demonstrated that mTORC1 disruption using raptor siRNA or RAD001 failed to inhibit 4E-BP1 phosphorylation in AML. Moreover, RAD001 failed to inhibit eIF4F assembly, to decrease the proportion of polysome-bound c-Myc mRNA, and to reduce the translation-dependent accumulation of oncogenic proteins. We identified the Pim-2 serine/threonine kinase as mainly responsible for 4E-BP1 phosphorylation on the S65 residue and subsequent translation control in AML. Our results strongly implicate an mTORC1-independent deregulation of oncogenic proteins synthesis in human myeloid leukemogenesis. Direct inhibition of the translation initiating complex thus represents an attractive option for the development of new therapies in AML.

Remission induction chemotherapy induces in vivo caspase‐dependent apoptosis in bone marrow acute myeloid leukemia blast cells and spares lymphocytes

The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response. We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment. We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability. More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction.

The effects of high dose methylprednisolone on apoptosis in children with acute lymphoblastic leukemia.

Rapid leukemic cell kill at initial diagnosis of patients with acute lymphoblastic leukemia (ALL) has been shown to be associated with a favorable outcome. The aim of the present study was to investigate the effect of high dose methylprednisolone (HDMP) on in vivo blast cell apoptosis in children with ALL. Annexin V-binding and Fas (CD95), Fas ligand (FasL; CD95L), and Bcl-2 expression in PB blasts were determined in newly diagnosed children with ALL before and 4, 24, 96 h after initiation of HDMP treatment (n=20) or conventional dose steroids (CDS) (n=10) as the control group. A decrease in absolute blast count (from 40.8 x 09 to 21.4 x 109/l) associated with an increase in apoptosis (14.2 to 26.9%) (P < 0.05) was detected 4 h after initiation of HDMP. A significant increase in Fas and FasL expression was detected 96 h after HDMP. There was no significant change in apoptosis, Fas and FasL expression from baseline in the control group treated with CDS. The changes in Bcl-2 expression after treatment was not significant in both groups. The results of this preliminary study have shown that HDMP treatment was effective in inducing immediate (within 4 h) blast cell apoptosis. The contribution of Fas/FasL interaction in the rapid component of cell kill remains to be determined, as the increase in the expression of these molecules was evident later.

Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells.

Procaspase 3 is a constitutive proenzyme that is activated by cleavage during apoptosis. The resulting enzyme is able to cleave several target proteins after the second aspartate of a DEVD sequence common to all the substrates of caspases 3 and 7 (DEVDase). Because active caspase 3 is a common effector in several apoptotic pathways, it may be a good marker to detect (pre-)apoptotic cells by flow cytometry (FCM). Materials and Methods Apoptosis was induced in U937 or bone marrow mononuclear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM). Viable and apoptotic cells were sorted by FCM on the basis of either fluorescein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVDase activity was measured in sorted populations by spectrofluorometry. Cleaved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-activated caspase 3 antibodies and analyzed by FCM. DEVDase activity was detected in sorted viable CAM- and DNR-treated U937 cells, demonstrating that a partial caspase activation occurred earlier than phosphatidyl-serine exposure and mitochondrial membrane potential dissipation. The presence of a low amount of active caspase 3 in the treated viable cells was confirmed in situ with PE-conjugated anti-active caspase 3 antibodies. The same antibody was used in combination with FITC-annexin V and CD45-PC5 to study caspase 3 activation in acute leukemia blast cells after in vitro DNR and IDA treatment. Both anthracyclines induced a caspase 3-dependent apoptosis that was more efficient in blast cells than in contaminating lymphocytes. These results demonstrate that anti-active caspase 3 labeling can be an alternative to fluorogenic substrates to efficiently detect early apoptosis by FCM in heterogeneous samples. They also confirm that anthracyclines induce blast cell apoptosis by a caspase 3-dependent pathway.

Effects of retinoids on cell toxicity and apoptosis in leukemic blast cells from patients with non-M3 AML

All- trans retinoic acid (ATRA) induces complete remission in acute promyelocytic leukemia (APL or M3). In this study we measured the effect of retinoids alone and in combination with daunorubicin (DNR) on cell growth and apoptosis in blast cells from patients with non-M3 AML. Cells from 21 patients were incubated in 0.2 μM daunorubicin for 1 h or in 1 μM ATRA or 9- cis-RA continuously and in the combinations of DNR with both retinoids. Cell toxicity and apoptosis were analyzed after 96 h. Both ATRA and 9- cis-RA reduced the viability significantly to 86 and 84%, respectively ( P=0.003 for ATRA and 0.02 for 9- cis-RA). The expression of CD34 correlated to a higher sensitivity to ATRA ( P=0.003). When retinoids were added to DNR the mean decrease in viability was 11 percentage points with ATRA ( P=0.003) and nine percentage points with 9- cis-RA ( P=0.02). Apoptosis was induced by both retinoids and the percentage of apoptotic cells was increased from 16% in the controls to 24% with ATRA ( P=0.03) and to 26% with 9- cis-RA ( P=0.04). When the retinoids were added to DNR the apoptotic rate increased from 41% with DNR alone to 51% with ATRA ( P=0.01) and to 49% with 9- cis-RA ( P=0.03). We conclude that ATRA and RA exert a slight but clear cytotoxic and apoptotic effect on AML blast cells after 96 h incubation and that retinoids can have an additive or synergistic effects on celltoxicity when added to daunorubicin.

Apoptosis and acute lymphocytic leukemia in children.

We examined in vivo spontaneous and prednisolone-induced apoptosis in peripheral blood samples of 23 children with ALL by flow cytometric and morphologic methods. There was no significant spontaneous apoptosis before the therapy. Six hours after prednisolone therapy, increased apoptosis was found in 19 of 23 cases. In one case, the apoptosis of blast cells could not be compared with the clinical data, because the patient died in sepsis during the induction therapy. In 18 of 22 evaluable cases, the in vivo apoptosis correlated with the decrease of leukemic blasts during the first 8 days of prednisolone monotherapy. In 20 of 22 children, a correlation was found between in vivo prednisolone-induced apoptosis and clinical outcome. The p53 gene expression was elevated in 2 of 10 patients. No elevation in the expression of bcl-2 gene was observed. In 6 of 23 cases, the glucocorticoid receptors were measured. The correlation of clinical responsiveness and gcR mRNA shows less parallelism than does the apoptosis correlation.