Smith, B. Douglas

Abstract Introduction: Topoisomerase I is a nuclear enzyme that relaxes torsional strain in DNA by nicking one DNA strand, allowing rotation, then resealing the DNA backbone. This is an important enzyme in replication and transcription, and therefore has been targeted by anti-cancer agents such as camptothecins. These agents intercalate into DNA at the topoisomerase I active site and inhibit the religation step catalyzed by the enzyme, causing increased topoisomerase I-DNA complexes that impede advancing replication forks or transcription complexes, leading to DNA damage and cell death. Platinating agents also stabilize topoisomerase I-DNA covalent complexes. The topotecan/carboplatin combination has recently shown promise in combination with the PARP inhibitor veliparib in aggressive and transformed myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (K Pratz et al., Clin Cancer Res. 23:899, 2017). Given that both classes of agents have a narrow therapeutic window, it is important to try to predict the sensitivity/resistance of these agents in malignancies such as acute myeloid leukemia (AML) and transformed MPNs. Some of mechanisms of drug resistance that have been described are decreased numbers of drug-stabilized topoisomerase I-DNA complexes due to drug efflux, diminished topoisomerase I expression or topoisomerase I mutation. Thus, being able to detect and quantify these DNA-protein complexes may provide insight into drug sensitivity. Methods: Using a unique monoclonal antibody that detects topoisomerase I-DNA covalent complexes (A. Patel et al., Nucleic Acids Res. 44:2816, 2016), immunofluorescence techniques were used to quantify these topoisomerase I-DNA complexes in AML cell lines (SET2, HEL, K562, U937, ML2, MV-4-11, THP1, HL60, ML1, and Molm13) and clinical AML samples. To verify the fidelity of the anti-topoisomerase I-DNA covalent complex antibody (TopIcc), siRNA knockdown methods were utilized with confirmatory immunofluorescence and Western blotting. Slot blots were additionally used to assess the presence of these complexes in AML cell lines. Western blots were performed to assess topoisomerase I levels in AML cell lines. Topotecan drug sensitivity analysis was performed by using an MTS assay. Results: With treatment of the topoisomerase I inhibitor, topotecan, there was an increase in the percentage of leukemia cells positive for the topoisomerase I-DNA covalent complexes. The degree of increase in complexes varied across cell lines and patient samples. To ensure we were detecting TopIcc by immunofluorescence, we performed a topoisomerase-I siRNA knockdown to confirm the specificity of the antibody for TopIcc's. Slot blots on multiple AML cell lines confirmed the presence of topoisomerase-DNA covalent complexes. The Western blot of all the AML cell lines, ordered from low to high TopIcc revealed increasing amounts of topoisomerase I. The MTS assay revealed lower topotecan IC50 values in three of the cell lines with higher complex formation, including Molm13, ML1 and ML2 (IC50 6-8 nM compared to 13-46 nM in the other cell lines). Conclusions: Based on recent clinical results, there has been renewed interested in topoisomerase I inhibitors, especially for aggressive and transformed MPNs. Topoisomerase I-DNA complex repair/downregulation has been a hallmark of drug resistance to topoisomerase I inhibitors. Therefore, detecting and quantifying these complexes in treated samples has been hypothesized to provide insight into the drug sensitivity, which is particularly important in camptothecins and platinating agents given their narrow therapeutic windows. Immunodetection techniques using an anti-TopIcc antibody may help predict sensitivity of AML to topoisomerase I inhibitors and will be used as a correlative biomarker study in an upcoming phase II randomized study of topotecan/carboplatin with or without veliparib or placebo in advanced myeloproliferative disorders and chronic myelomonocytic leukemia (ClinicalTrials.gov NCT03289910). Disclosures Pratz: AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy; Millenium/Takeda: Research Funding. Kaufmann:Mayo Clinic: Patents & Royalties: Co-inventor on a patent held by Mayo Clinic for the use of the anti-TOP1cc antibody as a diagnostic reagent.

Abstract Background: CXCR4 is a chemokine receptor over-expressed on > 75% of cancers, including myeloid leukemia blasts. Ulocuplumab (BMS- 936564) is a first in class, fully human IgG4 monoclonal antibody which inhibits the binding of CXCR4 to CXCL12, resulting in the mobilization of leukocytes from the bone marrow to the peripheral blood. Ulocuplumab also induces apoptosis of CXCR4+ cells in clinicial samples (i.e., leukemia blasts) and in in vitro experiments (e.g. in Tregs). It is thus hypothesized that Ulocuplumab may induce the mobilization and apoptosis of myeloid blasts & immunosuppressive cells which could improve the overall response rate to chemotherapy Objective: To conduct a first-in-man phase I dose escalation/ expansion trial to determine the safety, pharmacology, and clinical benefit of Ulocuplumab in relapsed/refractory AML Results: Seventy three subjects (median age, 58 yrs; range, 21-79 y) with relapsed/refractory AML were treated with Ulocuplumab and MEC [mitoxantrone, 8 mg/m2; etoposide , 100 mg/m2; and cytarabine, 1 g/ m2; i.v. d 1-5]. Thirty subjects in escalation received a single infusion of Ulocuplumab (doses of 0.3, 1, 3, or 10 mg/kg) one week prior to starting MEC and 3 additional weekly doses per MEC cycle thereafter (starting on Day 1). Ulocuplumab was escalated to a maximum of 10 mg/kg without any dose-limiting toxicity during monotherapy or in combination with MEC in the 1st cycle. In expansion phase, 43 patients were similarly treated with10 mg/kg Ulocuplumab and MEC. The overall complete remission and complete remission with incomplete blood count recovery rate (CR/CRi) was 51%. Subjects with first CR > 6 months had better ORR (16/23, 70%) than those with CR1 ≤6 months or primary induction failure (6/20, 30%). Of note, four subjects had CR/CRi after a single dose of Ulocuplumab monotherapy. Transient, mild/moderate thrombocytopenia was the only treatment-related AE documented with Ulocuplumab monotherapy. Only one subject presented a mild infusion reaction on Cycle 1 Day 1. The safety profile in combination with MEC was similar to MEC alone as was the 60 day all-cause mortality (16.3%). A median 2- and 5-fold mobilization of leukocytes and leukemic blasts into the peripheral circulation was reported at day 8, respectively. There was a trend demonstrating that higher CXCR4 expression on AML blasts correlated with a positive clinical response. Reversible and manageable hyperleukoctosis occurred in one subject. Conclusions: This study shows that the blockade of the CXCR4-CXCL12 axis with Ulocuplumab has antileukemic activity and safely improves the historic response rate achieved with MEC alone (i.e.,24-28%). Disclosures Becker: Bristol-Myers Squibb: Research Funding. Off Label Use: Etoposide is indicated in the management of refractory testicular tumors and small cell lung cancer. . Foran:Bristol-Myers Squibb: Research Funding. Altman:Bristol-Myers Squibb: Research Funding. Yacoub:Bristol-Myers Squibb: Research Funding. Castro:Bristol-Myers Squibb: Research Funding. Sabbatini:Bristol-Myers Squibb: Employment. Dilea:Bristol-Myers Squibb: Employment. Wade:Bristol-Myers Squibb: Employment. Xing:Bristol-Myers Squibb: Employment. Gutierrez:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Smith:Bristol-Myers Squibb: Research Funding.

Abstract Abstract 1531 Checkpoint kinase (Chk1) is a serine-threonine kinase that is activated via phosphorylation in response to DNA damage and is critical to the regulation of cell cycle progression. Ara-C triggers sequential activation of ATR kinase and Chk1 ex vivo to induce the S phase slowing that accompanies Ara-C treatment. Inhibition of Chk1 may abrogate S phase slowing, thereby preventing repair of Ara-C-induced DNA damage and potentiating the antitumor activity of Ara-C. The Hsp90 inhibitor tanespimycin (17-AAG) has been shown to enhance the cytotoxicity of Ara-C in part through Chk1 downregulation (RA Mesa et al., Blood 2005). While a phase I trial of Ara-C plus 17-AAG resulted in decreased blast cell Chk1 phosphorylation, drug toxicities precluded the clinical use of tanespimycin (SH Kaufmann et al., Haematologica, in press 2011). We have now tested the selective Chk1 inhibitor SCH 900776 (SCH 776) in combination with Ara-C in a Phase I dose-escalation trial in 24 adult patients with relapsed and refractory acute leukemias. AML (21) ALL (2) CML-BC (1) Male/Female 9/12 1/1 1/0 Age (range) 57 (23–73) 30, 63 36 Previous Ara-C 19 0 1 Relapsed 8 1 0 No. Prior CRs 2 (1–3) 2 0 Prior Stem Cell Transplant 6 0 0 Refractory 13 1 1 No. Prior Regimens 2 (1–4) 3 3 Prior Stem Cell Transplant 1 0 0 Secondary AML 8 0 0 MDS/MPD 6 0 0 Treatment-Related 2 0 0 Adverse Genetics 13 1 1 Single cytogenetics 3 1 0 Complex cytogenetics 11 0 1 Patients received Ara-C 667 mg/m2 on days 1–3 and 10–12 via 72-hour IV infusion along with SCH 776 on days 2, 3, 11, and 12 over 15 min IV starting at 10 mg/m2 (n=3) escalated to 20 mg/m2 (n=3), 40 mg/m2 (n=6), 56 mg/m2 (n=6), and flat dose of 140 mg (n=6) administered over 30 min IV. Maximal administered dose was achieved at 140 mg due to grade 3 prolonged QTc interval (n=1) and grade 3 hand-foot skin reaction (n=1). None of the expected mucosal or marrow toxicities of timed sequential Ara-C were exacerbated by SCH 776. Complete tumor clearance from day 14 bone marrow was detected in 13/24 (54%). CR plus CRi occurred in 7 patients (29%), with 6 of those CRs occurring at 40 mg/m2 or higher (response rate 40% for ≥ dose level 3). Examination of sequential samples of marrow blasts harvested pretreatment (day 0), 24 hours after initial Ara-C infusion prior to SCH 776 (day 2), and 2 hours after the second SCH 776 administration (day 3) demonstrated increased phosphorylation of Chk1 at Ser317 or Ser345 beginning at 40 mg/m2 consistent with the presence of unrepaired damage and further ATR activation. Pharmacokinetic (PK) data in each cohort showed lack of significant accumulation of SCH 776 in plasma. We plan for a randomized Phase II trial with 100 mg flat dose (or equivalent of 56 mg/m2) of SCH 900776 to assess any contributions to net Ara-C cytotoxicity and efficacy in relapsed and refractory acute leukemias. Disclosures: Gore: Celgene: Consultancy, Equity Ownership, Research Funding. Loechner:Merck Research Laboratories: Employment. Horowitz:Merck Research Labs: Employment, stock. Karp:Schering Merck: Research Funding.

Abstract Abstract 400FN2 Patients with primary and secondary myelofibrosis (MF) have a worse prognosis than those with essential thrombocytosis (ET) or polycythemia vera (PV). MF and chronic myelomonocytic leukemia (CMML) share chromosomal and genetic lesions such as ASXL1 and JAK2 mutations, but effective treatments are lacking. The aim of the study was to identify perturbed pathways in MPN and CMML that were amenable to targeted molecular therapies. In a large genomic analysis of MPN cases, we identified recurrent deletions of multiple DNA repair genes, including BRCA2 and Fanconi anemia (FANC) proteins, which have been associated with poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity. Neoplastic cells from these patients are hypersensitive to PARP inhibitors in vitro and may provide a therapeutic strategy to improve treatment response for patients with advanced MPNs. Genomic screens utilizing SNP-A karyotyping were performed on primary cells from 144 patients with either ET, PV, or MF (90 were previously reported, McDevitt et al., ASH 2009). We focused on the potential functional relevance of recurrent genomic deletions, including BRCA2, by performing a GO analysis using the EASE software application (Hosack et al. 2003). This revealed statistically significant (p≤0.05) enrichment of 13 biological pathways. Notably, genes involved in cell cycle regulation and DNA repair (DR) were enriched in MF loss regions (p=0.007) as compared to regions lost in ET and PV. Several genes directly involved in DR including BRCA2, XRCC2, and several members of the FANC pathway, also critical for DNA crosslink repair, were deleted or present in copy neutral LOH regions. DNA methyltransferase inhibitors are being tested clinically in MF patients, and both cell cycle and DNA repair genes have been identified as epigenetically silenced in hematological malignancies. We therefore screened for BRCA1, BRCA2, FANC-C, L, and N promoter CpG island methylation in a series of 48 MPN samples. No methylation was identified in BRCA2 and FANC members, suggesting a potential haploinsufficiency mechanism. Methylation of BRCA1 was identified in a small number of cases and is under further study in a larger cohort to investigate the effects on BRCA1 expression, DR and sensitivity to PARP inhibitors. Based on these genetic and epigenetic findings, we then focused on a functional analysis of the homologous recombination (HR) pathway. Leukocytes from normal controls and MPN patients were exposed to ionizing radiation (IR) and stained for Rad51 foci, a marker of HR activity. Six of 15 MPN/CMML samples (40%), but none of the controls failed to form IR-induced Rad51 foci, suggesting impaired HR. Of the samples that demonstrated defects in HR [MPN-U, MF, CMML (2), MDS/MPN-U, and CML], there was no significant correlation between the MPN subtype, the JAK2 mutation status, or the specific metaphase karyotype, but the most sensitive case in our series exhibited a deletion of Ch13q that spanned the BRCA2 gene. We then examined sensitivity of MPN vs. normal myeloid progenitors to the PARP inhibitors veliparib and/or olaparib in methylcellulose (MC)-based colony forming assays. Failure to form Rad51 foci with irradiation was statistically correlated with PARP inhibitor sensitivity (p = 0.021). When an expanded series of cases were then analyzed for PARP inhibitor sensitivity using the MC colony assay, lower IC50s for both veliparib and olaparib were observed in MPN cases (N= 25, IC50S=2.69uM vs. 11.2uM, N=15 IC50S=.64uM vs. 1.27uM, respectively) relative to normal controls (p =.0003, p =.05, via Wilconon rank-sum test respectively). Two cases in this series were treated on a clinical trial combining the ABT-888 PARP inhibitor with salvage chemotherapy consisting of carboplatin and topotecan (Karp et al., ASH 2010). Both patients, one with sAML evolving out of PPV-MF, and a second with accelerating MPN-U, had failed four prior therapies but achieved complete clearance of bone marrow blasts at day 14. Interestingly, cells from this MPN-U patient (13q-) were the most sensitive to PARP inhibition in vitro. Additional accrual and correlative evaluations are underway and will be updated at the meeting. Collectively, these results demonstrate that a subset of MPN and CMML patients have intrinsic genetic and epigenetic defects affecting the FANC and HR pathways, providing pre-clinical rationale for the use of PARP inhibitors in the treatment of aggressive MPN and CMML. Disclosures: No relevant conflicts of interest to declare.

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 BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%. The major reason for treatment failure in AML is resistance to chemotherapy. Thus, there is an urgent need for identification of novel therapeutic agents for AML. Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS). Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death. MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA. MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death. Whether MTH1 inhibitors have any activity against AML is not known. METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours. In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588. Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry. To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml). Percentage apoptosis was calculated by normalizing to vehicle only control. With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls. Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control. RESULTS: TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL. In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC50 3.1-21.3µM, Figure 1). Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death. In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTrackerLow cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC50 6µM, Figure 3). Analysis of additional primary AML samples is ongoing. DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells. MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells. In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine. Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Disclosures No relevant conflicts of interest to declare.

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.

Abstract On behalf of the ECOG-ACRIN Cancer Research Group Background: Venous thromboembolism (VTE) occurs in 2-12% of patients with acute leukemia (AL) despite disease and therapy-associated thrombocytopenia, and can be associated with significant morbidity and mortality. Due to limited high-quality studies, there are no evidence-based guidelines for VTE prophylaxis in this patient population. Based on our experience and the lack of consensus guidelines, we hypothesized that there would be a wide range in provider practice regarding methods of VTE prevention in patients with AL. To determine the current individual practices in North America, we devised a web-based survey of VTE prophylaxis practice among clinicians caring for patients with AL. Methods: An anonymous 19 question web-based survey, approved by the Johns Hopkins Institutional Review Board, was distributed by email to members of the ECOG-ACRIN Cancer Research Group on 10/22/14. Four reminders were sent at two-week intervals with the survey closing on 12/8/14. Results: Of the total 215 respondents, 64 were excluded for the following reasons: 52 did not directly manage medical care of AL patients, 3 referred AL patients to other centers, 5 left blank responses to all questions regarding VTE prophylaxis, 1 was a duplicate entry, and 3 described their position as support or office staff. One hundred fifty-one responses were eligible for analysis, with a response rate of 20.9% among physicians who treated leukemias. One hundred forty-seven were from the United States and 4 from Canada, representing 88 different institutions and 37 states or provinces. Overall, 47% and 45% of providers reported using pharmacologic VTE prophylaxis during induction and consolidation phases, respectively. Among the providers using pharmacologic prophylaxis, 60% designated 50,000/μL as the platelet count threshold below which they would hold prophylaxis and 26% used a platelet count of 30,000/μL. Fewer providers chose a platelet count of 20,000/μL (4%), 75,000/μL (4%), and 100,000/μL (2%). Lastly, 2% held prophylactic anticoagulation only in the setting of an active bleed and 2% reported variable thresholds. Approximately 15% of providers did not utilize any VTE prophylaxis while 36% used mechanical methods and ambulation. Among providers who did not recommend pharmacologic prophylaxis, the most commonly cited reasons were the perceived high risk of bleeding (51%), absence of data supporting use (38%), and perceived low risk of VTE (11%). Conclusions: These results demonstrate wide variation in clinician practice regarding VTE prevention in hospitalized AL patients during induction and consolidation therapy. Our findings emphasize the need to develop standardized, evidence-based guidelines in this at-risk population. VTE: venous thromboembolism, AL: acute leukemia, SC: subcutaneous, TED: thromboembolic deterrant stockings, SCD: sequential compression device Figure 1. Practice patterns of VTE prophylaxis for AL patients during induction and consolidation therapies Figure 1. Practice patterns of VTE prophylaxis for AL patients during induction and consolidation therapies Disclosures Barbarotta: Celgene, BMS, Novartis: Speakers Bureau. Prebet:CELGENE: Research Funding. Gore:Celgene: Consultancy, Honoraria, Research Funding.

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 788 FLT3 mutations are common in AML and are associated with poor prognosis. Lestaurtinib is a multi-targeted kinase inhibitor with potent activity against FLT3. In an attempt to definitively establish whether or not there is clinical benefit for FLT3 inhibition in the relapse setting, we conducted a randomized trial of chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse. Patients were randomized to receive either chemotherapy alone, consisting of mitoxantrone, etoposide, and cytarabine (MEC) or high-dose cytarabine (HiDAc) depending on the duration of CR1) or chemotherapy followed by 80 mg lestaurtinib orally BID. Patients on the chemotherapy only arm were eligible for crossover if they were refractory to therapy. Patients on the lestaurtinib arm received the drug for up to 112 days on study (they could continue to receive the drug through a separate extension protocol). The primary objective was complete remission (CR/CRp). Secondary objectives included overall survival (OS), safety, and tolerability. From January 2004 through December 2008, 224 patients at 52 different centers were enrolled and randomized, with 220 patients actually receiving therapy. Blood samples from patients on the lestaurtinib arm were obtained at baseline, on Day 15, and on Day 42 for pK studies and for determination of in vivo FLT3 inhibition and measurement of plasma FLT3 ligand (FL) and alpha-1 acid glycoprotein (AGP) levels. The median age for all patients enrolled was 55 years (53.5 in the control arm, 58.5 in the lestaurtinib arm), with 88% having FLT3/ITD mutations, 8% having D835 mutations, and 4% having both. The duration of CR1 was less than 6 months in 47% of patients in each arm. On the lestaurtinib arm, 107 patients received at least 1 week of lestaurtinib (mean 60.1 days, range 8-418), with 17 of these patients receiving drug on the extension protocol. On the chemotherapy only arm, 7 patients crossed over to receive lestaurtinib, and 31 patients went on to receive lestaurtinib on the extension protocol after outcome evaluation. Lestaurtinib was generally well tolerated following chemotherapy, with 104 Grade 3/4 adverse events in the lestaurtinib arm versus 99 in the control arm. There were 61 serious adverse events in the lestaurtinib arm versus 49 in the control arm. Early death (by Day 42) occurred in 22 lestaurtinib patients (2 progressive leukemia, 14 infectious, 6 organ failure) and16 control patients (6 progressive leukemia, 5 infectious, 5 organ failure). By intention to treat analysis, there were 29 CR/CRp in the lestaurtinib arm and 23 in the control arm (26% versus 21%; p = 0.35), with no difference in OS (4.73 months versus 4.57 months) between the two arms. Blood samples from 79 of the 107 lestaurtinib-treated patients obtained on Day 15 were analyzed for FLT3 inhibitory activity (plasma inhibitory activity [PIA] assay), as well as FLT3 ligand (FL), lestaurtinib, and AGP concentrations. Mean lestaurtinib plasma concentration was 12 uM at Day 15 and fell to 7.5 uM on Day 42. Mean lestaurtinib concentration at Day 15 was 13.3 uM in patients achieving CR/CRp and 11.5uM in non-responders. A target of >85% inhibition of FLT3 maintained at trough (pre-dose) was defined from previous studies. Of the 79 patients tested, 46 (58%) achieved this degree of FLT3 inhibition on Day 15. FL concentrations rose from baseline (15.6 pg/mL) to Day 15 (1148 pg/mL), and AGP concentrations rose by an average of 52% over the same period, both of which may have reduced the degree of FLT3 inhibition. Of the 46 patients with target FLT3 inhibition on Day 15, 18 (39%) achieved CR/CRp, while only 3 (9%) of the 32 patients with below target FLT3 inhibition achieved CR/CRp. Thus, in patients with FLT3 mutant AML at first relapse, pharmacokinetic factors and possible physiologic factors (FL and AGP) limit lestaurtinib's ability to effectively inhibit FLT3. FLT3 inhibition by lestaurtinib, when achieved, correlates with better CR rates, but in this trial that benefit was negated by a poor CR rate in those patients for whom the drug did not reach the target level of FLT3 inhibition. Overall, lestaurtinib treatment following re-induction chemotherapy failed to increase response rates or prolong survival of patients with FLT3 mutant AML in first relapse. Disclosures: Levis: Cephalon: Clinical Advisory Board member. Ravandi:Cephalon: Honoraria, Member, clinical advisory board, Research Funding. Erba:Cephalon: Research Funding, Speakers Bureau. Baccarani:Novartis Pharma, Bristol Myers Squibb, Merck Sharp & Dome, Pfizer: Consultancy, Speakers Bureau. Stone:Cephalon: ad hoc consultancy; Novartis: Research Funding, ad hoc consultancy. Advani:Cephalon: Research Funding. Douer:Cephalon: Honoraria, Research Funding, Speakers Bureau. Litzow:Cephalon: Research Funding. Tremmel:Cephalon: Employment, Equity Ownership. Bensen-Kennedy:cephalon: Employment. Smith:Cephalon: Research Funding.