Karp, Judith E.

Abstract Abstract 970 The mammalian target of rapamycin, mTOR, is a highly conserved serine/threonine kinase known to play a role in regulating mRNA translation, cell cycle progression, cell proliferation and apoptosis. As a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, mTOR is a component of two distinct complexes, TORC1 and TORC2. While TORC1 facilitates cell cycle progression from G1 into S phase by phosphorylating p70S6 kinase and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), TORC2 catalyzes the activating phosphorylation of Akt on Ser473, providing a feedback loop for further activation of mTOR. Phase II trials have shown activity of the TORC1-selective inhibitor rapamycin and its analogs in a wide range of lymphoma subtypes. The purpose of this study was to evaluate the anti-proliferative and pro-apoptotic effects of the dual TORC1/TORC2 inhibitor OSI-027 in human neoplastic lymphoid cells in vitro. MTS assays demonstrated that OSI-027 inhibited proliferation in a wide range of lymphoid lines, including SeAx (Sezary syndrome), DoHH2 (large cell lymphoma), RL (follicular lymphoma) and Jurkat (T cell ALL), as well as clinical lymphoma and T cell ALL samples, with IC50 values ranging from 0.078 to 10 μM. Propidium iodide staining followed by flow cytometry for subdiploid cells revealed induction of apoptosis within 48 h of treatment with OSI-027 (but not rapamycin) in SeAx, DoHH2, and Jurkat cells. Examination of Jurkat variants with alterations in key proteins involved in the death receptor versus mitochondrial pathway revealed diminished apoptotic responses to OSI-027 when Bcl-2 was overexpressed or caspase 9 was silenced, indicating involvement of the mitochondrial pathway. Immunoblotting for Bcl-2 family members revealed upregulation of Bim and Puma after a 48-hour exposure to OSI-027 but not rapamycin. This upregulation was also seen at the mRNA level, with a 12- to 20-fold increase in Puma mRNA and 4- to 12-fold induction of Bim mRNA. Small interfering RNA (siRNA)-mediated knockdown of Bim and Puma significantly diminished the apoptotic response to OSI-027. Because the Foxo3a transcription factor has been implicated in Bim and Puma expression and is known to be activated when Akt is inhibited, we next examined whether Bim and Puma induction was Foxo3a-dependent. Luciferase reporter assays showed that OSI-027 activated the full-length Puma and Bim promoters and that this activation was diminished when the Foxo3a binding sites were deleted or mutated. In addition, OSI-027 induced nuclear translocation of Foxo3a, while Foxo3a siRNA diminished OSI-027-induced apoptosis in Jurkat cells. Collectively, these results indicate that OSI-027 inhibits proliferation and induces apoptosis in a wide range of neoplastic lymphoid cells through a process that involves Foxo3a-mediated upregulation of Bim and Puma. These results also suggest that dual inhibition of TORC1 and TORC2 may be an effective treatment strategy in lymphoid malignancy. Disclosures: Barr: OSI Pharmaceuticals: Employment. Witzig:Novartis and Celgene: Patents & Royalties, Research Funding, Served on advisory boards with Novartis and Celgene – both uncompensated with compensation to Mayo Clinic.

Abstract Background: Cytosine arabinoside (ara-C) remains the backbone of most regimens for the treatment of AML. The incorporation of ara-C into DNA activates checkpoint kinase 1 (Chk1) causing replication fork slowing, the accumulation of cells in S phase, and diminished ara-C cytotoxicity. The selective Chk1 inhibitor MK-8776 abrogates ara-C-induced S-phase arrest, thereby enhancing ara-C-induced apoptosis in vitro.1 A phase I trial demonstrated the safety of MK-8876 in combination with infusional ara-C and correlative studies demonstrated increased ara-C-induced H2Ax phosphorylation in AML cells, consistent with unrepaired DNA damage.2 Herein, we report data from a phase II trial comparing ara-C plus MK8876 with ara-C alone. Methods: Patients with relapsed or refractory AML were randomized to receive either ara-C 2 gm/m2 via continuous infusion (CIV) over 72 hours on days 1 and 10 with MK-8776 100 mg flat dose infused over 30 minutes on days 2, 3, 11, and 12 (combo); or ara-C alone on the same schedule (control). The primary end point was response rate (CR+CRi). Adverse events (AEs) for all patients were reported according to the NCI-CTCAE version 4.0. Correlative studies were performed on blood and marrow samples collected prior to treatment on days 1, 2 and 3 and at one hour after MK-8776 infusion (PB only). Marrow and circulating blasts were analyzed using semi-quantitative flow cytometric determinations of blast cell gH2AX expression, a marker of DNA damage. Results: Thirty-two patients were treated at 4 sites with 14 assigned to the combo arm and 18 to the control arm. The study was stopped early due to lack of drug availability. The median age was 60 (range 29-72) and 17 (53%) were male. Nine (28%) had refractory AML, 16 (50%) were in first relapse, and 7 (22%) were in second relapse. Eleven (34%) had undergone a prior bone marrow transplant (BMT). Risk stratification classified 1 (3%) patient as favorable risk disease, 11 (34%) as intermediate-1, 8 (25%) as intermediate-2, and 11 (34%) as adverse risk. There were 5 (36%) responses (CR/CRi) and 1 (7%) partial response (PR) in the combo arm, and 8 (44%) responses and 1 (6%) PR in the control arm. Median survival did not differ significantly between the two groups (5.9 months in the combo arm vs. 4.2 months in the control arm). Sub-group analysis showed increased responses in the combo arm for the patients in first relapse (50% vs. 40%) and those with a prior BMT (50% vs. 29%). Most non-hematologic grade 3/4 treatment-related AEs were similar in the two groups including febrile neutropenia (64% vs. 61%), infectious complications (50% vs. 50%), and sepsis (21% vs. 17%). Patients in the combo arm experienced more QT prolongation (21% vs. 0%), hypertension (21% vs. 0%), and GI toxicities (21% vs. 0%); while those in the control arm had increased renal (0% vs. 11%) and liver toxicity (7% vs. 17%). Two deaths were attributed to treatment, and both were in the control arm. Eight combo patients had serial PB samples and 2 had serial marrow samples studied by flow cytometry. There was an increase in the percentage of gH2AX+ CD34+ cells on Day 2 directly after MK-8876 infusion (16.9% prior and 36.4% at one hour), which occurred to a lesser extent on Day 3 after MK-8876 infusion (12.7% prior and 15.6% at one hour). Three of eight patients demonstrated a sustained increase in gH2AX+ CD34+ from Day 2 to Day 3, and all three achieved either a PR or CR. Conclusion: This is the first phase II study building on prior work suggesting that inhibition of Chk1 would increase response to ara-C in patients with relapsed or refractory AML. The study confirmed that treatment with the combination is feasible with similar toxicities between the treatment and control groups. The response rates were similar between the two groups with improved responses in patients receiving the combo in first relapse and following BMT. Correlative studies support the finding that responding patients show a sustained increase in DNA damage and this may serve as a biomarker in future studies. Chk1 remains a potential target to improve treatment outcomes with ara-C. References: 1. Schenk EL et al. Clin Cancer Res. 2012 Oct 1; 18(19):5364-73. 2. Karp JE et al. Clin Cancer Res. 2012 Dec 15; 18(24):6723-31. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 3276 PARP is activated in response to DNA single strand (SS) breaks and is pivotal to the base excisional repair pathway for chemotherapy-damaged DNA. The orally bioavailable PARP inhibitor V delays DNA repair and potentiates the cytotoxicity of multiple classes of chemotherapy drugs including topoisomerase I inhibitors and platinating agents in leukemia cell lines. A previous clinical trial of T+C yielded promising results in adults with refractory acute leukemia at the maximum tolerated dose (MTD) of T 1.6 mg/m2/d + C 150 mg/m2/d by intravenous continuous infusion (IVCI) × 120 hrs. We are conducting a Phase I dose-escalation trial of V given orally twice daily Days 1–8 with T+C given by 120 hr IVCI Days 3–7. Acute Myelogenous Leukemia (AML, 33 patients) Acute Lymphoblastic Leukemia (ALL, 4 patients) Median Age (range) 570 (40-75) 55 (42-62) Relapsed 8 No. Prior CRs 2 (1-3) Prior Stem Cell Transplant 2 0 Refractory 25 4 No. Primary Refractory 10 1 No. Prior Regimens 3 (1-4) 3 (1-4) Prior Stem Cell Tranpslant 3 1 Secondary AML 12 Anecedent Hematologic Dx 6 Treatment-Related 6 Cytogenetics Intermediate 10 2 Adverse: Single/Complex 7/16 2 Ph+ (1 with complex) BRCA-1/BRCA-2 1 (mutation)/ 1 (13q14 deletion) 0 Deaths due to myocardial infarct and splenic infarcts with ascites occurred in one pt each when C 150 mg/m2 was added to V 10 mg BID and T 1.3 mg/m2, resulting in adjustment of the dose escalation schema for C and T. Of 27 pts treated at doses of V 10–20 mg orally BID Days1-8 and T 1.0–1.2 mg/m2/d + C 120–150 mg/m2/d for 120 hrs IVCI Days 3–7, 8 (30%) have experienced > grade 3 non-hematologic toxicity, 4 (15%) have had grade 4 toxicity and 1 (4%) died from sepsis with multi-organ failure. Overall response rate is 22% (2 CR, 1 CRi, 3 PR), with 4 in relapsed AML, 1 each in refractory AML and refractory ALL. Pharmacokinetic (PK) studies in pt plasma, marrow supernatant, and marrow blasts demonstrated that V does not accumulate with multiple dose administrations in plasma but does in marrow, is not altered by T or C administration, and is detectable in marrow supernatant and cells. Nuclear staining for phosphorylated histone H2AX (γH2AX) demonstrated that V 10 mg BID increased DNA damage by > 2-fold in 11/22 (50%) on Day 1 and 13/18 (72 %) on Day 4 after 24 hrs of T+C. A PAR ELISA demonstrated that V suppresses PAR levels to <50% of pretreatment values in 21/29 (72%) blood and 9/13 (69%) marrow samples. The MTD has not been reached and dose escalation is ongoing. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2742 Poster Board II-718 Background: The BCR-ABL inhibitor imatinib has revolutionized the treatment of CML, and improves the efficacy of chemotherapy in Ph+ ALL. Acquired imatinib resistance (IR) is an emerging problem. Patients who develop IR have a dismal prognosis and novel therapeutic strategies are needed. Mechanisms of resistance include mutations and overexpression of BCR/ABL. In addition, BCR/ABL-independent pathways may contribute to resistance. Mounting evidence suggests that aberrant histone deacetylase (HDAC) expression plays a key role in acquired IR. HDAC inhibition has been shown to downregulate BCR/ABL expression via multiple mechanisms. The functional effects of HDAC inhibition include cell cycle arrest and induction of mitochondrial damage and caspase-dependent apoptosis, both in cells that are imatinib sensitive (IS) and IR. We hypothesized that HDAC inhibition will demonstrate synergistic activity in combination with imatinib in IR cells. To test this hypothesis, we employed entinostat (SNDX-275), a novel, class I HDAC isoform selective inhibitor currently being tested in multiple phase 2 clinical trials. We explored the use of SNDX-275 alone and in combination with imatinib and dasatinib in IR and IS p210BCR/ABL expressing cell lines. Methods: K562 (p210BCR/ABL expressing CML cell line), IR K562 cells (Weisberg E, et al. Blood. 2000;95:3498), Ba/F3 cells, Ba/F3 cells transfected with p210BCR/ABL and Ba/F3 cells transfected with the T315I mutant form of BCR/ABL were treated with increasing concentrations of SNDX-275 (0.2 to 1.6μM), imatinib (0.1 to 1.6 μM), and dasatinib (0.3 to 250 nM) for 72 hours. The cells were also treated with combinations of SNDX-275 plus imatinib and SNDX-275 plus dasatinib at the same range of concentrations for 72 hours. All experiments were done in triplicate. Drug-related effects on cell viability were determined by MTT cytotoxicity assays. CalcuSyn software was used to calculate the IC50 for each drug in each cell line and to calculate the combination index (CI) for cell lines treated with combinations of imatinib or dasatinib plus SNDX-275. A CI from 0.9 to 1.1 indicates that the two drugs have an additive effect, below 0.9 indicates synergy between the two drugs and above 1.1 indicates antagonism. Results: In the Ba/F3 p210BCR/ABL and Ba/F3 T315I cells, SNDX-275 alone had minimal activity up to 1 μM. In K562 and IR K562 cells SNDX-275 alone led to significant cytotoxicity with an IC50 of 0.662 μM and 0.495 μM respectively. In the IR cell lines, cotreatment with SNDX-275 enhanced the activity of both imatinib and dasatinib. For the IR K562 cells, imatinib plus SNDX275 was potently synergistic with a CI of 0.6234. Dasatinib plus SNDX275 treatment of IR K562 cells was additive with a CI of 1.1015 (figure 1). Similarly, in the Ba/F3 T315I cells imatinib plus SNDX275 resulted in synergistic activity (CI = 0.87) and additive activity when treated with dasatinib plus SNDX275 (CI = 0.97). On the contrary, cotreatment of non-IR cell lines with dasatinib or imatinib plus SNDX-275 did not result in synergistic activity and revealed evidence of antagonism. In the K562 cells, the combination of imatinib plus SNDX-275 was antagonistic with a CI of 1.308 (figure 1A). In the Ba/F3 p210BCR/ABL cells, the combination of dasatinib plus SNDX-275 was strikingly antagonistic with a CI of 2.9. Conclusions: BCR/ABL-independent mechanisms of resistance, including aberrant acetylation of histone and non-histone proteins secondary to the deregulation of HDACs, contribute to IR in patients with CML and Ph+ ALL. Our data support this hypothesis, as the HDAC inhibitor SNDX-275 enhances the activity of imatinib and, to a lesser extent, dasatinib in IR p210BCR/ABL expressing cell lines. Therefore, SNDX-275 may represent a novel therapeutic approach to patients with IR CML and Ph+ ALL, and a phase I clinical study is planned. In contrast, SNDX-275 may antagonize the activity of the tyrosine kinase inhibitors in nonresistant CML and Ph+ ALL. To our knowledge, this is the first report of antagonism between a HDAC inhibitor and tyrosine kinase inhibitors in CML. Further investigation is necessary to define the mechanism and clinical significance of this antagonism. Disclosures: No relevant conflicts of interest to declare.

Abstract Among mechanisms underlying cytotoxic drug resistance is activation of diverse DNA damage response (DDR) pathways. Poly(ADP-ribose) polymerases (PARP)-1/2 facilitate both single- and double-strand break (DSB) repair and play a key role in the base excision repair (BER) of chemotherapy-damaged DNA. The PARP inhibitor veliparib (V) potentiates the cytotoxicity of different chemotherapeutics, including temozolomide (TEM). TEM induces distinct alkylating events in neoplastic cells whose ultimate outcome depends on the interaction of BER, mismatch repair (MMR), O(6)-methylguanine-DNA methyltransferase (MGMT), and DSB repair. While clinical activity of TEM has been mainly observed in MGMT-deficient AML, V potentiated cytotoxicity of TEM in leukemia cells in vitro in the setting of MGMT overexpression or deficient MMR pathway (Mol Cancer Ther, 2009). Methods: We conducted a Phase I study to determine maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of V+TEM, using a 3+3 dose escalation. Patients (pts) ≥60 years (yrs) with newly diagnosed poor cytogenetic-risk AML/ALL who were not candidates for intensive therapy, or ≥18 yrs with relapsed/refractory AML/ALL, secondary AML (therapy-related or arising from MDS or MPN), and CMMoL-2 were eligible. Any number of prior regimens, including allogeneic transplant (alloSCT), were allowed. V was given orally day (d)1 once, then twice a day on d4-12 at one of 6 dose levels (DL) (DL1A-B 20mg; DL2-DL3-DL4-DL5-DL6: 40-80-120-150-200 mg). TEM was given orally once a day on d3-9 (DL1A 150 mg/m2/d; DL1B-DL6 200 mg/m2/d). 28-day cycles (cy) were repeated depending on response/tolerability (4-6 weeks delay allowed) with V on d1-8 and TEM d1-5. TEM was taken on empty stomach with antiemetics and V was taken irrespective of meals. Results: Forty-nine pts with median age 69 yrs (range, 22-88; 47% ≥70) were treated. Of 47 AML pts, 29 (62%) had secondary AML and 27 (57%) adverse karyotype. Median number of prior treatments for AML was 1 (range, 0-6): 18 (38%) had median 1 prior therapy (range, 1-3) for MDS; 30 (64%), 9 (18%), 34 (69%) received hypomethylating agents, alloSCT and intensive chemotherapy, respectively. Overall 42 (85%) pts were refractory to their last treatment. Pts received a median of 1 (range, 1-7) cy of therapy. Two did not complete cy 1, pt withdrawal d5 and progressive fungal pneumonia d9 with death d15 of progressive disease (PD). The MTD/RP2D was defined at V 150 mg and TEM 200 mg/m2; 2 of 4 pts treated at V 200 mg and TEM 200 mg/m2 developed dose-limiting toxicity of grade (gr) 3 oral mucositis/esophagitis. The most frequent drug-related toxicities (NCI CTC v4) were gr 1/2 nausea/vomiting (39%), fatigue (26%), oropharyngeal mucositis (26%), constipation (12%), and diarrhea (10%). Other common toxicities were infectious, including febrile neutropenia (29%), pneumonia (20%), bacteremia (18%). One (2%) pt died ≤d30 and 12 (24%) ≤d60 mainly of PD (1 pt fungal pneumonia before count recovery d31). Overall response rate was 33% (complete remission (CR), hematologic improvement (HI)/stable disease) with 8 (16%) pts achieving CR (1 CRi). Median overall survival was 5.03 months, for all responders 11.58 months, and for CR pts 19.89 months (Fig 1). Responses occurred at all DLs. Three CR pts underwent alloSCT; 2 remain in CR at ~3 yrs. Pharmacokinetics (PK): V or TEM PKs were not altered with co-administration. There was a correlation between the DLT of mucositis and V single (Cmax P=0.005; AUC P=0.009) and multiple dose exposure (Cmax P = 0.02; AUC P=0.03). Pharmacodynamics and pharmacoepigenetics: Four of 39 pts examined had MGMT methylation (3 CR; 75%) and 2 had BRCA-1 methylation (1 HI) in peripheral blood (PB) or bone marrow (BM) mononuclear cells (MC). Defective FancD2 pathway was observed in the BMMC of 19/19 pts using FancD2 ubiquitylation assays but did not correlate with response. V reduced PAR levels by >75% in PBMC of most pts and in the presence of TEM. Induction of γ-H2AX in CD34+ cells was seen upon V/V+TEM treatment. Conclusion: V plus TEM demonstrated safety and activity in this resistant and elderly leukemia population. Response rate was higher in MGMT methylated pts, but responses were also seen in pts who had no MGMT methylation, had failed multiple therapies, had secondary AML and/or adverse karyotype. Future clinical study should aim to identify pts with defective DDR pathways who are most likely to respond to this therapeutic approach. Figure 1. Figure 1. Disclosures Off Label Use: Temozolomide is not approved for AML. Beumer:Millenium: Other: Research support. Gore:Celgene: Consultancy, Honoraria, Research Funding.

Abstract Abstract 1508 Background: Tipifarnib is an orally bioavailable farnesyltransferase inhibitor with well-established antileukemic activity in a minority of unselected patients with AML. Seeking to identify patients most likely to have tipifarnib-responsive AML, we applied a PCR-based quantitative 2 gene expression signature ratio (RASGRP1:APTX), which had been tested and validated retrospectively as a predictor of response and survival in previous large studies of tipifarnib, as a molecular screening tool for selection of patients to this pilot trial. Objectives: 1) primary: to determine the complete response (CR +CRi) rate in AML patients prospectively selected for tipifarnib treatment on the basis of a 2-gene signature (RASGRP1:APTX ratio) in bone marrow aspirates. 2) secondary: to assess safety, overall survival, and immunophenotypic expression of RASGRP1 on baseline bone marrow blasts for correlation with PCR-based detection. Methods: This was a multicenter, open-label, phase 2 study. Key eligibility criteria included 1) patients with previously untreated AML, age ≥65 years and deemed unsuitable for or refusing traditional induction chemotherapy, and 2) RASGRP1: APTX ratio of ≥5.0 (measured in bone marrow aspirate) based upon qRT-PCR. Other inclusion criteria included: ECOG PS 0–2, adequate end-organ function, and WBC < 30,000/microliter. Treatment consisted of Tipifarnib 300 mg BID × 21 days, with 7 days off. Dose escalation to 600 mg BID following cycle 1 was permitted for patients who had stable disease (SD) after cycle 1. Patients who achieved CR/CRi or PR after 2 cycles were eligible to continue treatment for up to 6 cycles maximum. Results: A total of 62 patients were consented to the trial. Of these, 21 (34%) were eligible based upon the required 2-gene ratio. Assay results were reported within 72 hours for all patients. Median age was 75 years. Fourteen (67%) patients had adverse karyotype and 14 (67%) had secondary AML (including 7 patients who received prior therapy for MDS). Three patients were replaced during cycle 1, due to withdrawal of consent or early unrelated death, such that 18 patients were considered evaluable for response. Median RASGRP1:APTX was 14.8. Two patients (11%) achieved CR and 6 (33%) additional patients had both SD and achievement of ≥50% reduction in bone marrow blasts by completion of cycle 2. Two of these 6 patients also had ≥50% increase in peripheral blood neutrophils or platelets. All 8 patients with CR or SD received at least 2 cycles of therapy. There was no correlation between baseline RASGRP1:APTX ratio and response. Overall, tipifarnib was well tolerated. Early death (≤ 30 days) was observed in only 1 patient (5%). The most common therapy-related non-hematologic toxicities (mainly grade 1–2) included: anorexia (33%), nausea (33%), fatigue (28%), febrile neutropenia (23%) and diarrhea (23%). Due to the trial not meeting its primary endpoint of at least 3 CR/CRi after 2 cycles, accrual to the trial was suspended. Conclusion: Monotherapy with tipifarnib in preselected elderly patients with AML and a RASGRP1:APTX ratio of ≥5 was associated with significant antileukemic activity and good tolerance. Although the primary endpoints during the first stage of the trial were not met, this study demonstrated the ability to rapidly and efficiently execute a biomarker-driven trial in an aggressive malignancy. Given the observed antileukemic activity in this selected population, along with a favorable toxicity and bioavailability profile for chronic dosing, further study of tipifarnib with less rigid endpoints than CR/CRi is warranted. Updated survival and RASGRP1 protein expression data will also be presented. Disclosures: Off Label Use: Tipifarnib in AML. Knoblauch:Johnson & Johnson: Employment. Karkera:Johnson & Johnson: Employment.

Abstract T is a farnesyltransferase inhibitor with clinical activity in elderly adults with newly diagnosed AML who are not candidates for TCC. For adults age ≥ 65 with poor-risk AML, T 600 mg BID for 21 out of 28–63 days induced complete remissions (CR) in 14%, median duration 7.3 mos, median survival 18.3 mos (Lancet JE, et al, Blood109:1387–1394,2007). In vitro, T+E exhibits synergistic anti-AML activity. In an attempt to increase CR rates in elderly AML pts, we conducted a Phase I trial of oral T+E, with escalation of both drugs and 14 vs. 21 days of T every 28–63 days (median 31 days, range 29–50). T+E resulted in a 25% CR rate (21/84 pts), duration 2 – 26+ mos, with best rates achieved with T dose ≥ 400 mg BID for 14 days (Karp JE, et al, Blood108:130a,2006). In the context of this trial, we have conducted longitudinal laboratory studies of changes in DNA damage and apoptosis in AML marrow blasts obtained from 84 patients prior to T+E (Day 0) and on Day 8 of T+E. Blasts were enriched by Ficoll density gradient separation and fixed in 70% ethanol. Persistent, unrepaired DNA damage was quantified by flow cytometry of cells stained with antibody to phosphorylated histone H2AX (γH2AX). Apoptosis was measured by flow cytometry of propidium iodide-stained cells with sub2n DNA content as observed in cell cycle distribution. To date, we have analyzed changes in in vivo γH2AX from 10 pts (4 CR, 6 stable disease or no response, SD/NR). CR pts had a 2-fold increase (range 1.91–2.22) in DNA damage induced by Day 8 of T+E relative to Day 0 values, while patients with SD/NR had no such increase (0–1.06) in DNA damage. Similarly, the apoptotic fraction of the cell cycle distributions of CR patients increase by 2-fold (range 1.2–5.3) for Day 8 marrow blasts. In contrast, changes in apoptosis were widely divergent for SD/NR patients (range 0.2–7.0). Taken together, these preliminary studies on AML marrow blasts obtained prior to and during T+E therapy suggest a possible linkage among induction of DNA damage, apoptotic response, and clinical outcome. Full analysis of all acquired specimens is ongoing.

Abstract The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in hematologic malignancies. Rapamycin analogues, which partially inhibit mTOR complex 1 (mTORC1), showed limited anti-tumor activity due to feedback mechanisms involving mTORC2 and incomplete inhibition of mTORC1. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR kinase domain. The purpose of this study was to delineate the mechanisms of mTOR dual inhibitor induced apoptosis in human neoplastic lymphoid cells in vitro. MTS assays and propidium iodide staining followed by flow cytometry for subdiploid populations demonstrated that OSI-027 and MLN0128 inhibited cell proliferation and induced apoptosis in a wide range of lymphoid cell lines including Jurkat, Nalm-6, Molt-4, and SeAX. Raptor and Rictor knockdown in Jurkat and Nalm-6 increased cell death, suggesting both mTOR complexes play a role in apoptosis. 4EBP1 phosphorylation was inhibited by mTOR dual inhibitors, but not by rapamycin. Expression of 4EBP1 T37A/T46A and 4EBP1 T37A/T46A/S65A/T70A, which mimic dephosphorylated 4EBP1, increased Puma mRNA and protein levels as well as apoptosis. Moreover, 4EBP1 knockdown abrogated mTOR dual inhibitor induced Puma upregulation and cell death, further supporting the role of 4EBP1 dephosphorylation in mTORC1 dependent apoptosis. In accord with the known dependence of c-Myc translation on the eIF4E/eIF4G complex, we also observed c-Myc downregulation after treatment with OSI-027, MLN0128 and 4EGI-1, but not rapamycin. Puma induction mirrored c-Myc downregulation under a variety of conditions, including expression of nonphosphorylatable 4EBP1 in parental Jurkat cells or wt 4EBP1 in 4EBP1 deficient cells. Furthermore, c-Myc knockdown induced Puma mRNA and protein as well as increased apoptosis. Collectively, these results support a model in which mTORC1 inhibition, acting through 4EBP1, induces Puma upregulation and apoptosis through c-Myc downregulation. In order to assess the parallel mTORC2-dependent Bim-mediated apoptotic mechanism, we utilized reporter assays and RNAseq experiments. OSI-027-induced Bim promoter activity decreased markedly when the nucleotides -29 to -18 were removed, suggesting that this response element is critical for OSI-027-induced promoter activation. In silico analysis identified eight transcription factors, including SP1, Egr-1, and Myb, that potentially bind this 12-bp region. In RNAseq experiments, we detected a 9-fold increase in Egr-1. Egr-1 upregulation was confirmed by qRT-PCR and immunoblotting after dual inhibitors treatment or Rictor knockdown. Moreover, dominant negative Egr-1 or Egr-1 knockdown diminished dual inhibitor-induced Bim promoter activation and Bim upregulation. Chromatin immunoprecipitation assays demonstrated that OSI-027 enhances binding of Egr-1 to a region of the Bim promoter including bp -29 to -18, further confirming that Egr-1 functions as a direct transcriptional activator for Bim upon mTOR dual inhibitor treatment. NFκB is a known transcription factor for Egr-1 and we observed increased p65 in the nucleus and increased NFκB transcriptional activity after dual inhibitor treatment. Overexpression of S32A/S36A IκB impaired the ability of dual inhibitors to induce NFκB transcriptional activation, Egr-1 mRNA and protein, Bim promoter activation and Bim mRNA and protein upregulation. Collectively, these results suggest that mTORC2 inhibition induces Bim upregulation and apoptosis through NFκB and Egr-1 transactivation. When fresh clinical ALL isolates were exposed to OSI-027 or MLN0128 ex vivo, inhibition of 4EBP1 phosphorylation along with upregulation of Egr-1 and Bim and/or c-Myc downregulation accompanied by Puma induction occurred, indicating that the pathways identified in ALL cell lines can also potentially be engaged in clinical ALLs. These observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors. Disclosures No relevant conflicts of interest to declare.

MLN4924, a novel chemotherapeutic agent designed to inhibit the Nedd8 Activating Enzyme (NAE), has shown promise in early clinical testing in relapsed and refractory Acute Myeloid Leukemia (AML). NAE inhibition prevents transfer of the Nedd8 protein to Cullin Ring Ligases (CRLs), leading to their inactivation and subsequent accumulation of the protein substrates they normally ubiquitinate and target for degradation by the proteasome. In malignant cells treated with MLN4924, various CRL substrates accumulate and induce a cytotoxic effect. Importantly, the substrates responsible for these effects vary among different malignancies depending on basal gene expression patterns. In order to shed light on the mechanism of MLN4924 anti-leukemic effects and identify potential mechanisms of resistance, the effects of MLN4924 on AML cell lines and primary AML samples were examined in vitro and in vivo. These studies identified c-Myc as the critical substrate governing the cytotoxic effect. In particular, we observed c-Myc transactivation of the PMAIP1 gene, leading to expression of pro-apoptotic protein Noxa, which functions to neutralize Mcl-1, activate Bak and Bax to allow release of cytochrome c from the outer mitochondrial membrane, and induce apoptosis. Importantly, MLN4924-induced Noxa upregulation was prevented by c-Myc shRNA; and MLN4924-induced killing was markedly diminished by Noxa siRNA. In further experiments, a synergistic cytotoxic effect was seen when MLN4924 was applied to AML cell lines in combination with agents that inhibit anti-apoptotic proteins other than Mcl-1, such as BH3 mimetics ABT-263 (inhibits Bcl-2, Bcl-xL, and Bcl-2) and ABT-199 (inhibits Bcl-2). Because expression of anti-apoptotic proteins within leukemia samples and between different AMLs is variable, and Mcl-1 upregulation often contributes to drug resistance and relapse in AML, this combination may be advantageous in several clinical settings. To follow up on the effects we observed with MLN4924 alone and in combination with BH3 mimetics, we have designed a multi-parameter flow cytometry based assay to assess response to these agents among hematopoietic stem cells (HSCs) and progenitor populations in primary leukemia bone marrow isolates treated ex vivo. Pilot studies conducted in mononuclear cell fractions containing normal HSCs and hematopoietic progenitors isolated from umbilical cord blood did not undergo apoptosis in response to MLN4924. Ongoing studies aim to specifically assess whether MLN4924 alone or in combination with other anti-leukemic agents induce apoptosis or alter expression of CD47 (an anti-phagocytic protein frequently upregulated in AML) in HSCs and hematopoietic progenitor cell populations within primary AML bone marrow isolates. As our understanding of how HSCs and hematopoietic progenitors contribute to AML initiation and progression advances, the need to target various populations will emerge and may vary depending on disease stage. It will be critical to ascertain whether HSCs and other populations are sensitive or resistant to agents such as MLN4924 and others in order to formulate and apply efficient targeted therapies. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 47 Background In adults with newly diagnosed, poor-risk AML we previously demonstrated that induction therapy with Flavopiridol in combination with Ara-C and Mitoxantrone (FLAM,) in a timed-sequential manner, yields complete remission (CR) rates of 67% and median disease-free survival (DFS) of 13.6 months. These data suggest that FLAM might improve outcomes relative to standard “7+3” in newly diagnosed AML with poor-risk features. Thus, we are conducting a phase II randomized trial comparing the CR rates with FLAM vs. 7+3 induction for all newly diagnosed adult patients with AML with intermediate and poor-risk features. We also previously identified a population of CD34+CD38− cells with intermediate aldehyde dehydrogenase (ALDH) activity that appears to represent a leukemic stem cell (LSC) population. We are examining the effects of FLAM vs. 7+3 in eradicating this LSC population and determining if persistence of LSC's predicts for relapse. Objectives The primary objective is to compare the rate of CR after 1 cycle of FLAM vs. 1 cycle of 7+3. The secondary objectives are to compare toxicities, survival rates, and presence or absence of minimal residual disease (MRD) after both induction regimens. Methods All newly diagnosed adult patients aged 18–70 without favorable risk features were randomized in a 2:1 fashion between FLAM and 7+3 at 90 mg/m2 of Daunorubicin. To achieve balanced randomization, patients were stratified by age, secondary AML and leukocyte count. CD34+ cell subsets were analyzed by flow cytometry for CD38 expression and ALDH activity by Aldefluor. The trial is ongoing. Results To date, 62 patients are evaluable for the primary end point. Demographics and results are depicted in table 1. Patient characteristics were similar in both arms, although there were more patients with poor risk features in the FLAM arm than 7+3. Overall grade > 3 toxicity was similar between both arms. However, there were 3 cases of grade > 3 tumor lysis syndrome (TLS) with FLAM, with 1 death, vs. 0 with 7+3. There were also 3 deaths with FLAM before day 60 vs. 0 with 7+3. CR rate with FLAM was 68% (28/41), with 1 cycle of 7+3 was 48% (10/21) (p = 0.17), and with 2 cycles (7+3 + 5+2) was 52% (11/21). CR rates based on specific risk factors are depicted in table 2. There was a trend toward improved CR rates in patients with adverse genetics with FLAM (p = 0.17). Putative AML LSC's were distinguished from normal hematopoietic stem cells based on ALDH activity. The persistence of putative LSC's in patients after therapy was highly predictive of subsequent clinical relapse. Conclusions Based on the current CR rate of 68%, FLAM holds promise as a therapeutic option for patients with newly diagnosed AML with intermediate and poor risk features. In this preliminary analysis, there is a suggestion that FLAM leads to higher CR rates in patients with adverse genetics than 7+3. Additionally, the detection of MRD consisting of a residual putative LSC population strongly portended subsequent relapse, even in patients without evidence of leukemia. Disclosures: Off Label Use: Flavopiridol (Alvocidib) is an anti-leukemia agent.