Apoptosis In Blasts Research Articles

FLT4 as a marker for predicting prognostic risk of refractory acute myeloid leukemia.

Treating patients with refractory acute myeloid leukemia (AML) remains challenging. Currently there is no effective treatment for refractory AML. Increasing evidence has demonstrated that refractory/relapsed AML is associated with leukemic blasts which can confer resistance to anticancer drugs. We have previously reported that high expression of Fms-related tyrosine kinase 4 (FLT4) is associated with increased cancer activity in AML. However, the functional role of FLT4 in leukemic blasts remains unknown. Here, we explored the significance of FLT4 expression in leukemic blasts of refractory patients and mechanisms involved in the survival of AML blasts. Inhibition or absence of FLT4 in AML blasts suppressed homing to bone marrow of immunocompromised mice and blocked engraftment of AML blasts. Moreover, FLT4 inhibition by MAZ51, an antagonist, effectively reduced the number of leukemic cell-derived colony-forming units and increased apoptosis of blasts derived from refractory patients when it was co-treated with cytosine arabinoside under vascular endothelial growth factor C, its ligand. AML patients who expressed high cytosolic FLT4 were linked to an AML-refractory status by internalization mechanism. In conclusion, FLT4 has a biological function in leukemogenesis and refractoriness. This novel insight will be useful for targeted therapy and prognostic stratification of AML.

Novel Compounds Synergize With Venetoclax to Target KMT2A-Rearranged Pediatric Acute Myeloid Leukemia.

In pediatric acute myeloid leukemia (AML), fusions involving lysine methyltransferase 2A (KMT2A) are considered hallmarks of aggressive AML, for whom the development of targeted specific therapeutic agents to ameliorate classic chemotherapy and obtain a complete eradication of disease is urgent. In this study, we investigated the antiapoptotic proteins in a cohort of 66 pediatric AML patients, finding that 75% of the KMT2A-r are distributed in Q3 + Q4 quartiles of BCL-2 expression, and KMT2A-r have statistically significant high levels of BCL-2, phospho-BCL-2 S70, and MCL-1, indicating a high anti-apoptotic pathway activation. In an attempt to target it, we tested novel drug combinations of venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor, in KMT2A-MLLT3, for being the most recurrent, and KMT2A-AFDN, for mediating the worst prognosis, rearranged AML cell lines. Our screening revealed that both the bromodomain and extra-terminal domain (BET) inhibitor, I-BET151, and kinase inhibitor, sunitinib, decreased the BCL-2 family protein expression and significantly synergized with venetoclax, enhancing KMT2A-r AML cell line death. Blasts t (6; 11) KMT2A-AFDN rearranged, both from cell lines and primary samples, were shown to be significantly highly responsive to the combination of venetoclax and thioridazine, with the synergy being induced by a dramatic increase of mitochondrial depolarization that triggered blast apoptosis. Finally, the efficacy of novel combined drug treatments was confirmed in KMT2A-r AML cell lines or ex vivo primary KMT2A-r AML samples cultured in a three-dimensional system which mimics the bone marrow niche. Overall, this study identified that, by high-throughput screening, the most KMT2A-selective drugs converged in different but all mitochondrial apoptotic network activation, supporting the use of venetoclax in this AML setting. The novel drug combinations here unveiled provide a rationale for evaluating these combinations in preclinical studies to accelerate the introduction of targeted therapies for the life-threatening KMT2A-AML subgroup of pediatric AML.

CSF1R Inhibition Combined with GM-CSF Reprograms Macrophages and Disrupts Protumoral Interplays with AML Cells.

Simple SummaryAcute myeloid leukemia is a blood disease whose long-term treatment is not satisfactory due to frequent (>50%) relapse despite complete initial remission. By adopting protumoral properties, macrophages in the leukemia cell microenvironment may promote resistance to treatment leading to relapse. Our goal was to assess whether macrophages in contact with leukemia cells have an impact on blast survival and sensitivity to chemotherapy. We observed that leukemia cells can educate macrophages to support their survival and proliferation and reduce their sensitivity to therapy, as long as the CSF1 receptor remains active and they are in close contact. By inhibiting the activity of the CSF1 receptor, in the presence of GM-CSF, we could modify the macrophage phenotype and increase blast apoptosis and sensitivity to treatment. Our results indicate that leukemia therapies should not only target blasts but also their microenvironment and specifically macrophages and their receptor for CSF1.Relapse is a major issue in acute myeloid leukemia (AML) and while the contribution of gene mutations in developing drug resistance is well established, little is known on the role of macrophages (MΦs) in an AML cell microenvironment. We examined whether myeloblasts could educate MΦs to adopt a protumoral orientation supporting myeloblast survival and resistance to therapy. Flow cytometry analyses demonstrated that M2-like CD163+ MΦs are abundantly present, at diagnosis, in the bone marrow of AML patients. We showed that myeloblasts, or their conditioned medium, polarize monocytes to M2-like CD163+ MΦs, induce the secretion of many protumoral factors, and promote myeloblast survival and proliferation as long as close intercellular contacts are maintained. Importantly, pharmacologic inhibition of the CSF1 receptor (CSF1R), in the presence of GM-CSF, reprogrammed MΦ polarization to an M1-like orientation, induced the secretion of soluble factors with antitumoral activities, reduced protumoral agonists, and promoted the apoptosis of myeloblasts interacting with MΦs. Furthermore, myeloblasts, which became resistant to venetoclax or midostaurin during their interplay with protumoral CD163+ MΦs, regained sensitivity to these targeted therapies following CSF1R inhibition in the presence of GM-CSF. These data reveal a crucial role of CD163+ MΦ interactions with myeloblasts that promote myeloblast survival and identify CSF1R inhibition as a novel target for AML therapy.

CXCR4 Inhibition with BL-8040 in Combination with Nelarabine in Patients with Relapsed or Refractory T-Cell Acute Lymphoblastic Leukemia / Lymphoblastic Lymphoma

Background: The chemokine receptor, CXCR4, mediates the trafficking and retention of hematopoietic stem cells to the marrow through its interaction with the chemokine, CXCL12. CXCR4 is the most highly expressed chemokine receptor on T-ALL cells, and preclinical data demonstrate that CXCR4 is critical for the growth and survival of T-cell acute lymphoblastic lymphoma (T-ALL). Genetic loss or pharmacologic blockade of CXCR4 signaling suppresses T-ALL cell growth and leukemia initiating activity in murine models. BL-8040 is a novel synthetic peptide antagonist of the chemokine receptor, CXCR4. Compared to other CXCR4 inhibitors, BL-8040 is a more potent mobilizer of normal HSCs with sustained occupancy and inhibition of CXCR4. We previously reported that BL-8040 potently suppresses T-ALL cell growth in xenotransplantation models of T-ALL. To test the efficacy of BL-8040 in T-ALL by conducting a multicenter, open label pilot study of BL-8040 in combination with nelarabine in patients with relapsed or refractory disease (ClinicalTrials.gov: NCT0276338). Methods: Adults with relapsed or refractory T-ALL/LBL, age ≥18 years and ECOG PS ≤2, were eligible for the study. A peripheral blood lymphoblast count ≤50,000/mm3 was required prior to starting treatment. During cycle 1, subjects received daily subcutaneous administration of BL-8040 1.5 mg/kg from Day 1 to Day 6 with nelarabine 1,500 mg/m2 intravenously over 2 hours on Days 2, 4 and 6. In subsequent 21-day cycles, BL-8040 1.5 mg/kg was administered daily on days 1-5 with nelarabine 1,500 mg/m2 given on days 1, 3 and 5. Up to 4 cycles of treatment was allowed. Results: Nine patients with median age of 29 years (range 20-75) have been enrolled in the study out of which including fout patients were in untreated 1st relapse, one in 2nd relapse, two with relapsed and refractory disease, and two primary refractory patient. Two patients had failed prior allogeneic hematopoietic cell transplant. The rate of complete remission is 56% (5/9) with all responses occurring after 1 cycle of treatment. Adverse events occurred in 2 subjects requiring dose modification. Consistent with the known mechanism of BL-8040, one subject developed G3 leukocytosis after BL-8040 administration with the peripheral WBC increasing from 5.3 to 273 h/mm3 with evidence of spontaneous tumor lysis requiring interruption of BL-8040 and leukapheresis. A second patient developed injection site pain requiring omission of the final dose of BL-8040. Peripheral blood analyzed at baseline and 24 hours after the first dose of BL-8040 (prior to nelarabine) showed the following: 1) sustained blockade of CXCR4 on leukemic blasts; 2) mobilization of leukemic blasts into the blood; and 3) no induction of leukemia blast apoptosis as measured by activated caspase 3 expression. Conclusions: BL-8040 can be combined with nelarabine in subjects with relapsed or refractory T-ALL with encouraging CR rates. Evidence of on target effects on CXCR4 has been observed in this study with sustained CXCR4 receptor occupancy at 24 hours after administration along with tumor mobilization of ALL blasts into the peripheral circulation. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Kadia:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Bioline RX: Research Funding; BMS: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Stock:Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria; UpToDate: Honoraria; Agios: Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Brammer:Verastem, Inc: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Bioniz Therapeutics, Inc.: Research Funding. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Chen:BiolineRx: Employment. DiPersio:Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; Magenta Therapeutics: Equity Ownership; NeoImmune Tech: Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Incyte: Consultancy, Research Funding; Karyopharm Therapeutics: Consultancy. OffLabel Disclosure: BL-8040 for ALL

The Selective Anti Leukemic Effect of BL-8040, a Peptidic CXCR4 Antagonist, Is Mediated By Induction of Leukemic Blast Mobilization, Differentiation and Apoptosis: Results of Correlative Studies from a Ph2a Trial in Acute Myeloid Leukemia

▪Background: Persistence of minimal residual disease (MRD) after induction, consolidation and salvage therapies is an independent poor prognostic marker in AML. The bone marrow (BM) microenvironment is a protective niche that promotes chemotherapy resistance of leukemia cells. This residual subset of chemotherapy-resistant cells present during remission eventually contributes to the leukemia relapse/persistence. Adhesion of AML cells to BM stroma is dependent on the CXCR4 receptor binding to its ligand CXCL12. High levels of CXCR4 expression correlate with poor survival in AML. It is postulated that blocking the CXCL12/CXCR4 axis will dislodge AML cells from the protective microenvironment and, when combined with chemotherapy, may improve the response to therapy. BL-8040 (BKT140) is a short peptide which inhibits the binding of CXCR4 to CXCL12, resulting in mobilization of leukemic blasts from the BM to the peripheral blood (PB). BL-8040 has strong affinity for the CXCR4 receptor with long receptor occupancy and as a single agent induces differentiation and apoptosis of leukemia cells in preclinical models. A clinical trial assessing the safety and efficacy of BL-8040 in combination with cytarabine (Ara-C) for the treatment of relapsed/refractory AML patients was recently completed (NCT01838395). Here we report results from correlative studies done on samples from patients who participated in this trial.Objective: To assess BL-8040 single agent anti leukemia effects in relapsed/refractory AML.Method: Patients received a once daily SC dose of BL-8040 monotherapy on days 1-2 followed by the same dose of BL-8040 plus Ara-C (1.5g/m2 for patients ≥60; 3g/m2 for patients <60) per day on days 3-7. Six dose levels of BL-8040 (0.5 - 2.0 mg/kg) were tested. PD parameters such as mobilization of leukemic blasts and induction of differentiation and apoptosis were assessed after monotherapy with BL-8040 using frequent PB sampling and BM aspirates at baseline and on day 3 prior to Ara-C administration.Results: 45 patients (including 3 receiving compassionate use BL-8040) with a median age of 61 yrs were treated. The majority of patients had poor risk disease with 24% secondary AML and 17% with prior allogeneic stem-cell transplantation. Most patients were heavily pretreated with 19% relapsing after a short first remission (CR1 ≤12 months), 17% had ≥2 relapses and 45% refractory to 1-2 inductions. The CR+CRi rate was 38% in subjects receiving BL-8040 dose ≥1.0 mg/kg (n=39). Ongoing follow-up (FU) of responding patients (expansion phase; N=19) shows median EFS of 9.3 months (range 4.3-12.8 months). At the time of this analysis 10/19 patients are alive with a FU range of 0.96 - 12.8 months. FACS analysis revealed that BL-8040 monotherapy triggered an average 4.3-fold increase of immature AML blasts (CD45+Low/CD34+/CD117+/HLA-DR+) in PB (i.e., mobilization). Response to treatment was associated with more robust mobilization of AML blasts following BL-8040 monotherapy (responders 9.5 vs non-responders 1.7 fold median). Preferential mobilization of AML blasts over normal cells (4.7 fold vs. 1.4 fold, respectively) was confirmed by FISH analysis in a subset of patients with informative cytogenetic abnormalities. Induction of granulocytic differentiation of immature leukemia progenitors (2.5 fold) in the BM following BL-8040 monotherapy was noted. Finally, induction of AML blasts apoptosis (40% increase) by BL-8040 alone was evident by FACS in day 3 (pre-araC) bone marrow biopsies.Conclusions: The data demonstrate that sustained blockade of the CXCR4-CXCL12 axis with BL-8040 is safe and well tolerated and when given in combination with Ara-C improves the response rate achieved historically with Ara-C alone. In addition, treatment with BL-8040 as a single agent rapidly and efficiently induces mobilization, differentiation and cell death of AML blasts. This selective effect on chemotherapy-resistant cells may be translated into reduction of residual disease arguing for incorporation into front-line trials and such studies are ongoing. DisclosuresForan:Millennium Pharmaceuticals, Inc.: Research Funding; medscape: Honoraria; karyopharm: Honoraria; pfizer: Honoraria; novartis: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding. DiPersio:Incyte Corporation: Research Funding. Peled:Biokine Therapeutics: Employment. Abraham:Biokine Therapeutics Ltd: Employment. Pereg:BioLineRx Ltd: Employment. Vainstein:BioLineRx Ltd.: Employment. Oberkovitz:BioLineRx Ltd.: Employment. Aharon:BioLineRx Ltd.: Employment. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Activity of OTX015 (MK-8628), a BET-Bromodomain Inhibitor, in Acute Myeloid Leukemia (AML) Progenitor Cells

CONTEXT: Eradication of leukemic progenitor cells, defined by functional assays such as long-term culture (leukemic long-term culture initiating cells [L-LTC-IC]) is the goal of therapy in AML. Bromodomain and ExtraTerminal (BET) proteins are epigenetic readers that regulate the expression of genes with super-enhancers, including CMYC. BET inhibitors (BETi) such as JQ1 induce proliferation arrest and apoptosis in murine models of AML, in human AML cell lines and primary blasts. Their activity in human leukemic progenitors has not yet been reported. OTX015 (MK-8626) is an orally available BETi that can be safely administered to patients with a continuous low-dose regimen (Dombret et al. Blood. 2014). Single-dose exposure to OTX015 induces gene expression modulation characteristic of bromodomain inhibition, including downregulation of CMYC and upregulation of HEXIM1, inhibiting the viability of AML cell lines, and inducing apoptosis in primary AML blasts (Coudé et al. Oncotarget. 2015). To address the activity of OTX015 on leukemic progenitors, we analyzed (A) the clonogenicity of AML cell lines and (B) the frequency of primary L-LTC-IC after repeated low-dose exposure to OTX015.METHODS: (A) Five AML cell lines (OTX015 IC50 60 - 10,000 nM) were studied: OCI-AML3, NOMO-1, HL-60, KG1a and K562. After 24h starvation, OTX015 or vehicle (DMSO) was added daily to the culture medium for 3 days at various concentrations. After 96h, cells were assessed for gene expression by RT-qPCR and seeded in methycellulose. Colonies were scored after 14 days. (B) Bone-marrow mononuclear cells (BMNC) from AML patients obtained at diagnosis after informed consent were cultured for three weeks in a niche-like hypoxic milieu shown to maintain leukemic stem cells (Griessinger et al. Stem Cells Transl Med. 2014). OTX015 200 nM or DMSO was added weekly. This concentration is in the range of trough concentrations achievable at the MTD of OTX015 in phase I trials. Residual leukemic cells were sorted and plated on methylcellulose. Colonies were scored after 14 days. The resulting L-LTC-IC frequency was reported relative to the number of BMNC initially seeded.RESULTS: (A) To dissect the effect of OTX015 on AML progenitors from that on the leukemic bulk, we determined for each cell line a maximal OTX015 concentration that could be administered repeatedly for 3 days without significantly impairing proliferation or viability (MTT) at day 4 of culture (referred as low-dose concentration). As expected, this target concentration, ranging from 50 to 500 nM, was lower in cell lines with low OTX015 IC50. This prolonged low-dose exposure to OTX015 recapitulated BETi-associated gene expression changes including CMYC downregulation and HEXIM1 upregulation in all cell lines, and significantly reduced clonogenicity compared to DMSO in 4/5 cell lines, but not in NPM1-mutated OCI-AML3 cells (IC50: 60 nM, target concentration 50 nM), despite modulation of CMYC and HEXIM1 expression. Overall, there was no correlation between the level of CMYC repression and clonogenicity. Transcriptome analyses are ongoing to identify gene expression changes specifically associated with inhibition of clonogenicity.(B) L-LTC-IC frequency after prolonged exposure to 200 nM OTX015 was determined in specimens from 11 AML patients with variable oncogenetics. L-LTC-IC frequency was reduced in 5/11 patients, reaching statistical significance in 3 cases; OTX015 reduced L-L-LTC-IC in 3 of 4 NPM1-mutated samples, but not in any of the 3 patients with high-risk cytogenetics. No clear correlation was found between induction of apoptosis on primary blasts after short-term, and L-LTC-IC reduction after long-term 200nM OTX015 exposure respectively. Patients' samples number is being extended to identify oncogenetic predictors of L-LTC-IC reduction.CONCLUSION: Our results suggest that in AML cell lines or primary samples, prolonged exposure to low concentrations of the clinically-available BET inhibitor OTX015 results in activity against leukemic progenitors independent of induction of proliferation arrest or apoptosis in blasts. Molecular mechanisms and oncogenic markers of this activity are being investigated. These results warrant clinical investigation of the anti-leukemic properties of prolonged low-dose OTX015 administration. DisclosuresRiveiro:Oncoethix: Research Funding; OTD: Employment. Herait:Oncoethix: Other: shareholder; Oncoethix: Other: Chief medical officer; Oncoethix: Other: shareholder. Dombret:Oncoethix: Research Funding. Itzykson:Oncoethix: Research Funding.

Pentoxifylline during steroid window phase at induction to remission increases apoptosis in childhood with acute lymphoblastic leukemia.

Pentoxifylline (PTX) has been shown to increase chemotherapy-induced apoptosis. A clinical trial was developed to evaluate the effect of the addition of PTX to the induction steroid window phase in children with acute lymphoblastic leukemia (ALL). Thirty-two children were enrolled on this study. Children with a new diagnosis of ALL were randomly assigned to receive prednisone (PRD) 40 mg/m(2)/day only during the 7-day treatment pre-phase (PRD group, 11 patients) or to receive PRD with PTX (10 mg/kg/day) (PTX group, 11 patients); the control group included children with normal bone marrow (10 patients). Bone marrow aspiration (BMA) was performed at diagnosis (day -7) in all groups, and at day 0 (end of PRD window) for patients with ALL (PRD and PTX groups). Apoptosis was evaluated by flow cytometry (FC) using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) stains. Statistical analysis was performed using the Mann-Whitney U test. Apoptotic index at day -7 was similar in all groups. However, at day 0 post-treatment, apoptosis was significantly higher in the PTX group than in the PRD group (p < 0.001). There were no serious adverse effects associated with PTX. PTX potentiates blast apoptosis induced by PRD in children with ALL during steroid window phase.

Antileukemic activity of sulforaphane in primary blasts from patients affected by myelo- and lympho-proliferative disorders and in hypoxic conditions.

Sulforaphane is a dietary isothiocyanate found in cruciferous vegetables showing antileukemic activity. With the purpose of extending the potential clinical impact of sulforaphane in the oncological field, we investigated the antileukemic effect of sulforaphane on blasts from patients affected by different types of leukemia and, taking into account the intrinsically hypoxic nature of bone marrow, on a leukemia cell line (REH) maintained in hypoxic conditions. In particular, we tested sulforaphane on patients with chronic lymphocytic leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, and blastic NK cell leukemia. Sulforaphane caused a dose-dependent induction of apoptosis in blasts from patients diagnosed with acute lymphoblastic or myeloid leukemia. Moreover, it was able to cause apoptosis and to inhibit proliferation in hypoxic conditions on REH cells. As to its cytotoxic mechanism, we found that sulforaphane creates an oxidative cellular environment that induces DNA damage and Bax and p53 gene activation, which in turn helps trigger apoptosis. On the whole, our results raise hopes that sulforaphane might set the stage for a novel therapeutic principle complementing our growing armature against malignancies and advocate the exploration of sulforaphane in a broader population of leukemic patients.

The Response of FLT3/ITD AML to FLT3 Inhibition: Apoptosis of Peripheral Blood Blasts and Differentiation of Bone Marrow Blasts

Abstract 943FLT3/ITD mutations are present in approximately 23% of adult AML cases. FLT3/ITD therefore presents an attractive therapeutic target for tyrosine kinase inhibitors. To date, the clinical responses to FLT3 inhibitors have been primarily limited to clearance of peripheral blood blasts, while bone marrow blasts remain largely unaffected. AC220 (quizartinib), a novel FLT3 inhibitor with 10–50 times more potency in vivo than other FLT3 inhibitors, has had a high response rate in an exploratory subset of patients in an ongoing clinical trial for relapsed and refractory FLT3/ITD AML. A high proportion of FLT3/ITD AML patients treated with AC220 displayed the typical rapid clearance of peripheral blasts seen with other FLT3 inhibitors. Strikingly, however, (as reported at last year’s ASH meeting), the bone marrow blasts underwent terminal differentiation in response to AC220 treatment over the course of 4–8 weeks, as established by the presence of the same FLT3/ITD mutation in mature circulating neutrophils isolated after treatment with AC220. This differentiation was frequently accompanied by a syndrome of fever, skin nodules resembling Sweet’s syndrome, and pulmonary nodules. To study the mechanisms associated with this phenomenon, we developed an in vitro model of FLT3 inhibitor-induced differentiation. In order to more closely reproduce the bone marrow microenvironment in vitro, we prepared human stromal cell layers derived from healthy bone marrow donors and co-cultured them with Molm14 cells (FLT3/ITD cell line), as well as with primary AML blasts from FLT3/ITD AML patients in the presence and absence of AC220. In the absence of co-culture with stroma, both the Molm14 cell line and the primary samples underwent rapid apoptosis in the presence of increasing concentrations of AC220, similar to the response of peripheral blood blasts in AC220 -treated patients. However, when grown in co-culture with stroma, Molm14 cells underwent relatively rapid (24–48 hours) morphologic differentiation, as evidenced by the development of characteristic multi-lobed nuclei, and by reduction of nitroblue tetrazolium (NBT) after stimulation with endotoxin (respiratory burst activity). Similar data were observed in Molm14 cells treated with the FLT3 inhibitor sorafenib, indicating that this differentiation is likely a class effect of FLT3 inhibitors. We have also used our model system to study differentiation in primary AML patient blasts. Pre-treatment blasts were obtained from a patient enrolled on the AC220 trial (who exhibited terminal differentiation of marrow blasts in response to AC220), and differentiation was induced in vitro by co-culturing the blasts with stroma in the presence of AC220. After five days of drug exposure during stromal co-culture, the blasts remained viable (assessed by trypan blue exclusion), but morphologically began to resemble myelocytes, and demonstrated reduction of NBT in response to endotoxin. Flow cytometry analysis of these blasts after eight days of AC220 exposure demonstrated increased side scatter, expression of CD15, and loss of CD117 and CD34, all indicative of maturation. Blasts cultured in the absence of stroma displayed decreased P-FLT3, P-STAT5, and P-ERK in response to AC220. In contrast, blasts exposed to AC220 on stroma demonstrated continued activity of P-ERK despite loss of P-FLT3 and P-STAT5. CEBPalpha protein levels were significantly higher in blasts on stroma, but decreased rapidly over the first 48 hours of culture. These findings confirm that a FLT3/ITD mutation can be sufficient to induce a differentiation block, and, in the bone marrow microenvironment, FLT3 inhibition does not induce immediate apotosis, but rather differentiation. The continued activity of P-ERK (presumably by parallel signaling pathways activated by stromal cells) may be responsible for the survival of the blasts in the setting of FLT3 inhibition, with CEBPalpha inducing a differentiation program in this context. We conclude, therefore, that the response of a FLT3/ITD AML patient to potent FLT3 inhibition consists of rapid apoptosis of blasts in the peripheral blood, and, in most cases a slower process of terminal differentiation in bone marrow blasts. Furthermore, our in vitro model will allow us to explore the precise mechanisms whereby differentiation is blocked in FLT3/ITD AML, and how this can be overcome with FLT3 inhibition and other targeted therapies. Disclosures:Borowitz:BD Biosciences: Research Funding. Levis:Ambit Biosciences, Inc: Consultancy.

Escalation Trial of Clofarabine Followed by Escalating Dose of Fractionated Cyclophosphamide in Adults with Relapsed or Refractory Acute Leukemias,

Abstract 3615 Background:The prognosis of patients with relapsed/refractory acute leukemias (RRAL) continues to be very poor, with a complete remission (CR) rate of ≤ 30% and a one-year disease-free survival of ≤ 10%. Consequently, there is no standard of care for the treatment of RRAL at the current time. Clofarabine (CLO) is a second-generation purine nucleoside analog that has shown activity against RRAL. The administration of CLO followed by Cyclophosphamide (Cy) enhances the DNA damage induced by Cy and increases AML/ALL blast apoptosis rates. We previously published a phase I study in RRAL patients in which a timed-sequential approach using CLO followed by Cy (days 1–3/8-10 or 1–2/8-9) resulted in promising clinical responses, but was associated with unacceptable toxicity (Karp, Blood 2007). Given the lengthy marrow aplasia, which was a dose-limiting toxicity (DLT), we conducted another phase I dose-escalation trial of CLO followed by escalating doses of fractionated Cy in adults with RRAL using an infusion schedule of 4 consecutive days (CLO-Cy × 4). Methods:One cycle of CLO-Cy was defined as 28 days. In order to assess the impact of CLO on Cy induced DNA damage, a 50% dose of CY was given alone on day zero and the remaining 50% was given after CLO on day 1. Thereafter, full dose Cy was given after CLO days 2–4. Full daily Cy doses ranged from 600–800mg/m2/day (total 2.4–3.2gms/m2). CLO was administered days 1 through 4 at 10 mg/m2/day for dose levels 1 and 2, 15 mg/m2/day for dose level 3, and 20 mg/m2/day for dose level 4. Results:Between 12/07 and 3/09, 26 adults (age 18 yrs) were entered on study. An additional 14 patients (pts) were enrolled between 4/09 and 3/10 in an expansion cohort determined by the phase 1 trial (dose level 1). 40 pts (28 AML [4 relapsed, 24 refractory], 12 ALL [3 relapsed, 9 refractory]) were given 45 cycles of CLO-Cy (5 pts received 2 cycles each). The average age was 48.5 years (range 22–73 years). The median number of prior regimens was 2 (range 1–5), including 6 who had prior allogeneic stem cell transplant. Of pts with AML, 21 (75%) had adverse cytogenetics, while 8 pts with ALL (67%) had adverse cytogenetics (2 with Philadelphia-positive ALL). The most common side effects were grade 3 and 4 hematologic toxicity. 6 pts (15%) died within 60 days of induction, 2 from septic complications and 4 from progressive disease. The average time to recovery of absolute neutrophilic count (ANC) of ≥ 500 was 40 days (range 31–78). 7 pts developed bacteremia and 2 developed fungemia. 10 pts developed grade 3 pneumonias, of which 7 were probably fungal. 5 pts developed pericardial/pleural inflammation or effusions (1=grade 3). 3 pts had reversible non-oligouric acute renal failure, but only one developed tumor lysis syndrome. Elevated liver function tests were noticed in 4 pts (2 grade 3). CLO-cytokine release syndrome (3 pts), hand-foot syndrome (3 pts), CLO-induced fever (1 pt), and mucositis (1 pt) were all ≤ grade 2. On day 14, 24 pts achieved complete tumor clearance, 4 pts had minimal disease, and 9 pts had residual disease. Of the 40 pts, 13 pts had an objective response (33%) with 11 CR, 2 PR. Of the 11 CR, 6 were in pts with non-adverse cytogenetics and 5 in pts with adverse cytogenetics. For AML, overall response rate (OR) was 25% (7/28) with 5 CR and 2 PR. For ALL, OR was 50% (6/12), all CR. At dose level 1 (total of 21 pts), OR was 48% (8 CR, 2 PR). Notably, the clinical responses were not dependent on the dose level. Among clinical responders, median pretreatment total white blood cell (WBC) was 3,310 (260–29,560) and median absolute lymphocyte count (ALC) was 1,737 [252–5948]. Median pretreatment IgG was 998 (226–1930), IgM was 67 (29–191), and IgA was 53 (<7–349). Median % decline in the ALC was 99%. The median % decline in IgG from D0 to D30 was 3%, for IgA was 4% and for IgM was 37%. Mean time to ALC recovery (ALC>200) was 47 days (32–85). Conclusions:This 4-day regimen of CLO followed by Cy demonstrated activity and tolerability in this heavily pretreated patient population with RRALs, with an OR of 33% (13/40). While the numbers are small, CLO-Cy may have particular activity in relapsed ALL. Immune reconstitution analysis showed profound and protracted decline in ALC without significant effects on IgG and IgA but perhaps with negative impact on early immunoglobulin responses (IgM). Further immunologic correlates should accompany clinical studies using CLO. Disclosures:Karp:Genzyme: Research Funding.

Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML

AbstractActivation of p53 by murine double minute (MDM2) antagonist nutlin-3a or inhibition of X-linked inhibitor of apoptosis (XIAP) induces apoptosis in acute myeloid leukemia (AML) cells. We demonstrate that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and down-regulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a–induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of second mitochondria-derived activator of caspases (SMAC) and by inducing the expression of caspase-6. Because both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

Treatment with Hsp90 Inhibitor, 17-AAG Overcomes Resistance to Small Molecule FLT3-Inhibitors in FLT3/ITD-Positive Leukemia Cells Harboring N676K-Mutation.

The receptor tyrosine kinase FLT3 is mutated in ~30% of acute myeloid leukemia (AML) and small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3-inihibitors have shown measurable clinical responses. However, most of these responses were transient and in some cases an additive single amino acid substitution (such as N676K) within FLT3 kinase domain was reported to be a possible cause for the temporary response. We previously showed a novel FLT3 inhibitor, FI-700, selectivity suppresses the growth of leukemia cells with FLT3 mutations (Kiyoi, et al., Clin Cancer Res 07). In order to examine how we can overcome drug resistance due to additional kinase mutations, we cultured MOLM-13 (FLT3/ITD-positive leukemia cell line) cells with increasing doses of FI-700 to generate several FI-700-resistant sublines. In 6 of 6 clones with resistance to more than 1 μM FI-700, FLT3N676K mutation was detected in one allele, and these were also resistant to small molecule FLT3-inhibitors such as AG1295 and AG1296. In a drug-screening for this kind of subline (MOLM-13/FLT3N676K), sub-molar treatment with molecular chaperon Hsp90 inhibitor, 17-AAG, inhibited growth as revealed by MTT-assays, and a combination of 17-AAG and FI-700 synergistically induced cell death as indicated by DNA-histograms and PI/Annexin-V assays. Expression of FLT3 by FACS and phosphorylation of FLT3 and its substrate molecules (such as STAT5), were significantly inhibited by the combined treatment of FI-700 and 17-AAG. To reveal the detailed mechanisms underlying these synergistic effects, we are currently investigating the binding of FLT3 and Hsp90, the degradation and the localization of FLT3 in these cells. We are also investigating the in vivo-effects of combined treatment in NOD/SCID mice inoculated with MOLM-13/FLT3N676K. These results imply that treatment of FLT3/ITD-positive leukemia cells with 17-AAG is a promising strategy for overcoming the drug resistance of cells with mutations in the FLT3 kinase domain.

The Level of Glycolytic Metabolism of AML Blasts May Predict Drug Sensitivity and Prognosis in Patients with AML

Solid tumors that show high levels of glycolysis are often refractory to therapies such as arsenic trioxide (ATO), which mediate their anti-tumor effect via increased mitochondrial free radical formation. We have previously shown that purely glycolytic, mitochondrial gene knock-out HL60r0 cells were significantly more resistant to apoptosis induced by combined ATRA+ATO treatment than non-glycolytic HL60 cells [Herst et al. Leuk Res 2008]. Here, we investigate whether the degree of glycolytic metabolism of AML blasts isolated from diagnostic bone marrow samples reflects in vitro drug sensitivity and duration of remission from AML. Following Ethics Committee approval, AML blasts from 22 patient bone marrow (BM) samples were isolated from bone marrow aspirates previously stored in the Peter MacCallum Cancer Centre tissue bank. On each sample of AML blasts we determined the level of glycolytic metabolism by % FCCP-inhibition of reduction of the water-soluble tetrazolium dye, WST-1/PMS at the cell surface [Herst, Biochim Biophys Acta, 2007] and compared results to those measured for several cell lines and 8 primary bone marrow samples of acute lymphoblastic leukemia (ALL). In samples where sufficient (>105) cells were available, we separately assessed the degree of blast apoptosis, via annexin V/propidium iodide staining and flow cytometric analysis, induced by a 72 hour culture in either 1mM all-trans retinoic acid (ATRA), 2mM ATO or combined 1mM ATRA+2 mM ATO. Analysis of glycolysis revealed that AML blast samples distributed into two non-overlapping groups (p=0.0001) of moderate (n=13) and high levels (n=9) of glycolytic metabolism. In contrast, the level of glycolytic metabolism in ALL blasts, normal donor peripheral blood T cells and several cancer cell lines (HL60, Hela, HeLaS3w, BW1100., EL4, A20) varied extensively (Figure 1A). Paired samples of both diagnosis and subsequent relapse BM were available from 3 patients with >80% blasts in both samples. In these paired samples the level of glycolytic metabolism did not alter (all moderately glycolytic at both time points) from diagnosis to relapse, suggesting that this is a stable metabolic feature of AML that is not modified, or selected by, exposure to prior chemotherapy. Highly glycolytic AML blasts were relatively resistant to combined ATRA and ATO treatment than moderately glycolytic blasts (p=0.025) but not to ATRA or ATO treatment alone (Figure 1B). Survival from the date of bone marrow sampling was also assessed and compared between high and moderate glycolytic cohorts. At the time of analysis, with a median follow up of 4 years, 6 out of 9 patients with highly glycolytic AML blasts remain alive. Conversely, all 13 patients with moderately glycolytic AML blasts have died of progressive disease, with median survival of 64 days (p=0.005 by Gehan- Breslow-Wilcoxon test). Our results suggest that the extent of glycolytic metabolism, as measured by % FCCP-inhibition of dye reduction may be used to identify chemo-refractory and chemo-sensitive subgroups of AML and may be potentially applicable in identifying patients who may benefit from treatment intensification or novel therapies. [Display omitted]

MEK Inhibitor AZD6244 Induces Cell Growth Arrest and Synergizes Nutlin-3a-Mediated Cell Death by Upregulating p53 and PUMA Levels in Acute Myelogenous Leukemia.

Raf/MEK/Erk signaling is constitutively activated in the majority of acute myeloid leukemias (AML), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and pro-apoptotic effects of AZD6244, a small molecule MEK1/2 inhibitor, as well as potential synergistic effects with the MDM2 antagonist Nutlin-3a in AML. AZD6244 inhibited proliferation of AML cells by arresting cells in G1 phase, which resulted from inhibition of cell cycle checkpoint proteins cyclin E and cdk2, and was associated with the modulation of cyclin D1, cyclin A, cyclin B1, p27 and Rb phosphorylation levels. AZD6244 also induced apoptotic cell death in AML cell lines with constitutive activation of MEK/ERK signaling (OCI/AML3, HL60 and MOLM13) via decrease of anti-apoptotic Mcl-1 protein expression and upregulation of pro-apoptotic Bax, Bak, Bad and Bim proteins. Next we tested the hypothesis that activation of p53 signaling with a small molecule MDM2 inhibitor Nutlin-3a would enhance the pro-apoptotic effects of MEK inhibition in AML. Combinations of AZD6244 and Nutlin-3a triggered profound apoptotic responses in AML cells with wild-type p53 (OCI/AML3, MOLM13 and Baf3-FLT3-ITD), with combination indices (averaged ED50=0.37, ED75=0.38, ED90=0.43) suggesting strong synergism. In contrast, no synergism was seen in p53 null cell HL60 (ED50=1.27, ED75=1.38, ED90=1.66) or in OCI-AML3 cells stably transduced with p53 shRNA. Mechanistically, combined blockade of MEK pathway and activation of p53 signaling resulted in marked upregulation of p53 expression and of the p53 target protein PUMA, while only partially suppressing Nutlin-induced p21 upregulation. This resulted in loss of the mitochondrial membrane potential, cytochrome c release and cleavage of caspase-9 and -3. Importantly, combined AZD6244 and Nutlin-3a treatment potently induced apoptosis in blasts from primary AML patients. In summary, AZD6244 induced growth-inhibitory and pro-apoptotic effects in AML cells with constitutive activation of MEK/ERK signaling. These effects were synergistically enhanced by simultaneous activation of p53 signaling by Nutlin-3a, resulting in the activation of the intrinsic apoptotic pathway. Altogether, these results strongly suggest potential activity of AZD6244 alone or in combination with MDM2 antagonists as novel mechanism-based therapeutic agents in AML therapy.

Increased TNF/NGF Receptor Expression on BCP-ALL Blasts Carrying the Prognostically Favorable TEL-AML Rearrangement.

TEL-AML the most common genetic alteration in childhood precursor B (BCP)-ALL is associated with a favorable prognosis yet the underlying mechanism remains largely unknown. Among the tumor necrosis factor (TNF) receptors CD40 is a key regulator of B cell maturation while nerve growth factor receptor (NGFR), CD27 and CD95 have been implicated in cell cycle arrest and blast apoptosis. Here we prospectively assessed baseline surface expression of the different TNF receptors and the costimulatory molecules CD70, CD80 and CD86 on primary BCP-ALL blasts in a pediatric cohort of TEL-AML positive (n=29) and negative patients (pts) (n=89) treated according to the CoALL06-97 protocol. In the TEL-AML positive subgroup, the percentage of blasts positive for CD40 (median; interquartile range: 100%, 100–100% vs. 82%, 56–97%; p<0.001), NGFR (36%, 11–77% vs. 8%, 1–49%; p=0.008) and CD27 (80%, 61–86% vs. 5%, 2–25%; p<0.001) was significantly higher than in the group negative for genetic aberrations such as TEL-AML, BCR-ABL and MLL-AF4. With low to negligible baseline expression for the remaining surface markers, no difference was detected for CD95 (p=0.24), CD70 (p=0.54), CD80 (p=0.87) and CD86 (p=0.06). As signaling via CD40 is known to modulate expression of costimulatory molecules of the TNF receptor/ligand family (CD27 and CD70) and of the IgG superfamily (CD80 and CD86), we compared upregulation of these molecules in TEL-AML positive and negative patients after CD40 crosslinking. To this end blast samples (n=31) were cultured for three days on genetically modified feeder cells expressing CD40 ligand. Although CD80 and CD86 expression was 5- and 3-fold (± 1 and 0.8 SEM) enhanced in CD40-activated blasts (p<0.001), there was no difference in TEL-AML positive and negative patients (CD80: p=0.13; CD86: p=0.17). In contrast there was a significant difference in CD70 upregulation with a 50-fold (± 42 SEM) increase of CD70 positive blasts in the TEL-AML group compared to only a 4.5-fold (± 0.7 SEM) upregulation in TEL-AML negative blasts (p=0.015). Of note as a costimulatory molecule, CD70 is critical not only for activation but particularly for expansion of cytotoxic T cells. CD40-induced upregulation of CD70 was accompanied by 2-fold (± 0.3 SEM) downregulation of initially high baseline expression of CD27 in the TEL-AML positive group only, a phenomenon that is also seen in maturation of normal CD27 positive B cells. In summary our findings provide first evidence for a differential phenotype of TEL-AML positive BCP-ALL blasts with regard to molecules of the TNF receptor/ligand family. Thus TEL-AML positive blasts exhibit a more mature immunophenotype with a significantly higher percentage of CD40, CD27 and NGFR positive blasts at diagnosis potentially contributing to superior survival by enhancing sensitivity towards pro-apoptotic signals via CD27 and NGFR and improving the T cell stimulatory capacity of BCP-ALL blasts by pronounced CD40-dependent upregulation of the costimulatory molecule CD70 critical for sustaining anti-leukemic immune responses.

Rapamycin stimulates apoptosis of childhood acute lymphoblastic leukemia cells

AbstractThe phosphatidyl-inositol 3 kinase (PI3k)/Akt pathway has been implicated in childhood acute lymphoblastic leukemia (ALL). Because rapamycin suppresses the oncogenic processes sustained by PI3k/Akt, we investigated whether rapamycin affects blast survival. We found that rapamycin induces apoptosis of blasts in 56% of the bone marrow samples analyzed. Using the PI3k inhibitor wortmannin, we show that the PI3k/Akt pathway is involved in blast survival. Moreover, rapamycin increased doxorubicin-induced apoptosis even in nonresponder samples. Anthracyclines activate nuclear factor κB (NF-κB), and disruption of this signaling pathway increases the efficacy of apoptogenic stimuli. Rapamycin inhibited doxorubicin-induced NF-κB in ALL samples. Using a short interfering (si) RNA approach, we demonstrate that FKBP51, a large immunophilin inhibited by rapamycin, is essential for drug-induced NF-κB activation in human leukemia. Furthermore, rapamycin did not increase doxorubicin-induced apoptosis when NF-κB was overexpressed. In conclusion, rapamycin targets 2 pathways that are crucial for cell survival and chemoresistance of malignant lymphoblasts—PI3k/Akt through the mammalian target of rapamycin and NF-κB through FKBP51—suggesting that the drug could be beneficial in the treatment of childhood ALL. (Blood. 2005;106:1400-1406)

Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia.

Mutations in the receptor tyrosine kinase FLT3 occur frequently in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Small molecules that selectively inhibit FLT3 kinase activity induce apoptosis in blasts from AML patients with FLT3 mutations and prolong survival in animal models of FLT3-induced myeloproliferative disease. A spectrum of structurally different small molecules with activity against FLT3 have been described, and their efficacy for treatment of AML and ALL is now being investigated in clinical trials. Here, we describe the results of an in vitro screen designed to identify mutations in the ATP-binding pocket of FLT3 that confer resistance to tyrosine kinase inhibitors. Mutations at four different positions (Ala-627, Asn-676, Phe-691, and Gly-697) were identified that confer varying degrees of resistance to PKC412, SU5614, or K-252a. FLT3 proteins mutated at Ala-627, Asn-676, or Phe-691 remained sensitive to higher concentrations of the inhibitors, but the G697R mutation conferred high-level resistance to each of these inhibitors as well as to six additional experimental inhibitors. These data provide insights into potential mechanisms of acquired resistance of FLT3 to small molecule inhibitors and indicate that the G697R mutation may be a clinically problematic resistance mutation that warrants proactive screening for additional inhibitors.

Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL.

The therapeutic and preventive activities of retinoids in cancer are due to their ability to modulate the growth, differentiation, and survival or apoptosis of cancer cells. Here we show that in NB4 acute promyelocytic leukemia cells, retinoids selective for retinoic-acid receptor-alpha induced an autoregulatory circuitry of survival programs followed by expression of the membrane-bound tumor-selective death ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand, also called Apo-2L). In a paracrine mode of action, TRAIL killed NB4 as well as heterologous and retinoic-acid-resistant cells. In the leukemic blasts of freshly diagnosed acute promyelocytic leukemia patients, retinoic-acid-induced expression of TRAIL most likely caused blast apoptosis. Thus, induction of TRAIL-mediated death signaling appears to contribute to the therapeutic value of retinoids.

Preferential induction of apoptosis of leukaemic cells by farnesol

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 μM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.