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Supplemental Figures S7. Supplemental Figures S7. Effects of JQ1 and OTX015 on BRD4 binding to MYD88 promoter region in DLBCL cell lines.

Purpose: In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-containing proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015.Experimental Design: We assessed the preclinical activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo experiments to identify the mechanism of action and the genetic features associated with sensitivity to the compound.Results: OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo experiments showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, especially with mTOR and BTK inhibitors. Gene expression signatures associated with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compound induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell–like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11.Conclusions: Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clinical investigation of OTX015 as single agent and in combination therapies. Clin Cancer Res; 21(7); 1628–38. ©2015 AACR.

Supplemental Figures S1-2. Supplemental Figures S1: effects of OTX015 on cell cycle and cell growth in DLBCL cell lines. Supplemental Figures S2: effects of OTX015 on apoptosis in DLBCL cell lines.

Supplemental Table S4: List of gene-sets associated with the sensitivity to OTX015.

Supplemental Figures S6. Supplemental Figures S6: OTX015 effects on the production of IL-4 and IL-10 in DLBCL cell lines.

Supplemental Figures S3-5. Supplemental Figures S3: the gene expression changes induced by OTX015 in DLBCL cell lines. Supplemental Figures S4: network of genes affected by OTX015. Supplemental Figures S5: similar biologic effects of OTX015 and JQ1.

Supplemental Table S1: Cell lines and growth medium.

The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.

Purpose: In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-containing proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015. Experimental Design: We assessed the preclinical activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo experiments to identify the mechanism of action and the genetic features associated with sensitivity to the compound. Results: OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo experiments showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, especially with mTOR and BTK inhibitors. Gene expression signatures associated with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compound induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell–like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11. Conclusions: Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clinical investigation of OTX015 as single agent and in combination therapies. Clin Cancer Res; 21(7); 1628–38. ©2015 AACR.

Our purpose was to determine, in patients with metastatic colorectal carcinoma treated with irinotecan single-agent after 5-FU failure, the most significant predictive parameters for tumour response, progression-free survival and toxicity. Between October 1992 and April 1995, 455 patients with 5-FU resistant metastatic colorectal carcinoma entered four consecutive phase II trials. The first two studies assessed tumour response, the other two were randomized studies which assessed the efficacy of racecadotril to prevent irinotecan-induced diarrhoea. Due to homogeneous main eligibility criterias, data from those studies could be pooled for statistical analysis. Potential clinical and biological predictive factors (PF) for toxicity, tumour growth control, e.g. response or stabilization and progression-free survival (PFS), were studied in multivariate analysis. 363 patients were evaluable for response, 432 were evaluable for PFS, 368 for neutropenia and 416 for delayed diarrhoea, respectively. Normal baseline haemoglobin level (Hb), time since diagnosis of colorectal carcinoma, grade 3 or 4 neutropenia or diarrhoea at first cycle and a low number of organs involved were the most PF for tumour growth control (P< 0.05). Significant prognostic variables for PFS were WHO Performance Status, liver and lymph-node involvement, time since diagnosis, age and CEA value (P≤ 0.02). Six groups of patients based on the number of unfavourable prognostic factors are presented. Baseline bilirubin, haemoglobin level, number of organs involved and time from diagnosis were PF for neutropenia; PS, serum creatinine, leukocyte count, time from 5-FU progression and prior abdominopelvic irradiation were PF for delayed diarrhoea (P≤ 0.05). These PF should help clinicians to anticipate for a given patient the probability to observe a response/stabilization or a toxicity. These results should also be prospectively confirmed in ongoing or future trials using irinotecan, both as a single agent and in combination with other drugs. © 2000 Cancer Research Campaign

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. Disclosures Riveiro: 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.

Background: OTX015 (OncoEthix SA, Switzerland) is a novel oral bromodomain and extraterminal (BET) protein family inhibitor, with in vitro and in vivo activity in a variety of hematologic and solid tumor cells. A phase Ib study with OTX015 in patients with hematologic malignancies is underway including a pharmacokinetic (PK) investigation. The PK objectives of this study were to determine the PK profile of oral OTX015 using a population approach. Materials and Methods: A multicenter, dose escalation study in cohorts of 3 to 6 patients with acute leukemia or other hematologic malignancies was performed with a dose escalation step followed by expansion cohorts at the recommended dose. Patients received oral OTX015 from 10 to 160 mg different schedules. PK blood samples from 7 time points were collected over 24 h post-administration on Day 1 for leukemia patients (complete PK) and 4 blood samples over 8 h post-administration for patients with other hematologic malignancies (limited PK). OTX015 plasma concentrations were measured using validated ultra-performance liquid chromatography with tandem mass spectrometry detection with a concentration range 1-250ng/mL. Analyses and population PK (PPK) modeling were performed with the nonlinear mixed effect modeling software program Monolix version 4.3. The following parameters were calculated absorption constant (Ka); apparent distribution volume (V/F); apparent clearance (CL/F) and lean body mass (LBM; calculated considering patient sex, weight and height) was considered as covariate. Results: 85 patients enrolled and treated from January 2013 to August 2014, randomized to six dose levels (10, 20, 40, 80, 120 and 160 mg) QD and 40 mg BID were evaluated. Among them, 81 patients with 630 plasma concentrations (607 + 23 BLQ) were evaluable for PK assessment. A 1-compartment open model adequately described the total OTX015 concentration-time curve. The PPK parameters obtained for the structural model were Ka = 0.74 h−1 (12%); V/F = 71.7 L (6.0%) and CL/F = 8.45 L/h (5.0%). The best correlation between OTX015 AUC values and dose was observed from 10 to 120 mg dose levels (R2 = 0.71). The absorption phase was linear and Tmax was between 1 and 4 h. Mean elimination half-life of OTX015 for all patients was 5.8 h (± 1.1). In the PPK study, the best descriptive model was obtained when LBM was considered in the analysis. A correlation between CL/F and V/F was also observed for OTX015. Conclusions: The PK of OTX015 is best described by a one-compartment model. Preliminary PPK analysis considering only the dose escalation cohort of the Phase I trial indicates that LBM is a good predictor of the OTX015 PK profile. This model should be validated in the ongoing expansion cohorts treated at 80mg QD. Citation Format: Elodie Odore, Francois Lokiec, Maria Eugenia Riveiro, Fabrice Bourdel, Carmen Kahatt, Patrice Herait, Esteban Cvitkovic, Keyvan Rezai. Pharmacokinetics of OTX015 in a phase Ib dose-finding study of patients with hematologic malignancies: Preliminary results of a population PK analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2015-4511

Background: OTX015 (Oncoethix) is a small molecule that specifically binds to BRDs 2, 3 and 4, thereby inhibiting binding to acetylated histones, resulting in downregulation of several oncogenes driven by super-enhancers. The therapeutic potential of BRD inhibitors has been demonstrated in several preclinical models including hematologic malignancies. Methods: The primary end point of this dose escalation study (3 + 3 design) was to establish the Phase 2 recommended dose (P2RD) and schedule in two independent cohorts of patients (pts) with either relapsed/refractory acute leukemia (AL) or other hematologic malignancies (OHM). OTX015 was given orally, daily (qd), with 1 bidaily (bid) dose level (DL) explored, continuously in OHM or for 14 days ON/7 days OFF in AL. Results: From January 2013 to August 2014, 41 pts with AL (AML 37; ALL 3) or high-risk myelodysplastic syndrome (HR-MDS 1) and 45 pts with OHM (22 diffuse large B-cell lymphoma [DLBCL], 11 other lymphomas, 12 multiple myeloma) were enrolled over 7 DLs from 10 to 160 mg. Dose-limiting toxicities (DLT) during the first treatment cycle were observed at doses >120 mg in AL (diarrhea, asthenia) and doses >80 mg in OHM (primarily thrombocytopenia). Additionally, both AL and OHM pts treated at >80 mg experienced grade 1-2 gastrointestinal events (diarrhea, dysgueusia) that hampered compliance, though not meeting DLT criteria. PK showed dose-proportional OTX015 exposure (AUC0-24h) up to 120 mg qd; trough plasma concentrations at 80 mg qd reached the GI50 cut-off of 500 nM for sensitive tumor cell lines in vitro. Two complete remissions (CR) and 1 CR with incomplete recovery (CRi) were observed in pts with relapsed/refractory HR-MDS or AML secondary to MDS and 2 CR and 1 partial response in DLBCL pts. OTX015 P2RD was determined as 80 mg qd with a 14 days ON/7days OFF schedule, based on kinetics of platelet nadir and recovery. Conclusion: OTX015 is an orally available molecule showing dose-proportional exposure up to 120 mg qd with a favorable tolerance profile, and evidence of clinical activity in refractory DLBCL and AML/HR-MDS. Expansion cohorts in AML secondary to MDS, de novo AML and DLBCL at the P2RD are ongoing.

Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models. Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software. Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells. Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. Figure 1: Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Figure 1:. Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Disclosures Riveiro: OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

Rationale: BET-bromodomain (BRD) proteins are DNA readers that bind acetylated histone (H) tails preferentially at hyperacetylated superenhancer promoter regions and trigger gene transcription. The expression of several oncogenes, including c-MYC, is epigenetically regulated by BRD. OTX015 is a BRD 2, 3 and 4 inhibitor that prevents BRD binding to acetylated H4 and downregulates gene expression of BRD-dependent genes. OTX015 has been shown to inhibit the growth of diffuse large B-cell lymphoma (DLBCL) cells in vitroand in animal models. Patients & Methods: Patients with non-leukemic hematologic malignancies refractory or resistant to standard therapies were enrolled in an ongoing phase 1 clinical study. Lymphoma patients had to have failed at least two lines of systemic therapy and have evaluable disease. OTX015 was given orally daily (QD) without a planned rest period, with 3-week cycles (cy). Successive cohorts of 3-6 patients were treated at increasing dose levels (DL) from 10 to 120 mg QD to determine the maximum tolerated dose (MTD) or the biologically optimal dose. A BID schedule was tested at DL 4 (40 mg x 2). Pharmacokinetics was assessed on day 1 and residual concentrations were measured on days 2, 8 and 15. Lymphoma assessment was performed according to Cheson’s criteria every 6-8 weeks. Results: From January 2013 to June 2014, 37 non-leukemic patients (18 DLBCL, 9 other lymphomas, 10 myeloma) were treated over 5 dose levels, 33 of whom were evaluable for dose limiting toxicity (DLT). Median age was 67 years (range 27-83), 22 patients were male, 27 patients had ECOG 0-1. Patients had a median of 4 prior therapy lines (range 2-8), including 10 patients with autologous stem cell transplantation. The median number of OTX015 cycles administered was 2 (range 1-10+). No DLTs were observed through DL4 (80 mg QD). Asymptomatic and rapidly reversible grade 4 thrombocytopenia was the DLT at DL4 BID (40 mg x2) and 120 mg QD continuous. Sixteen patients experienced grade 3-4 thrombocytopenia and 3 patients had asymptomatic grade 3-4 neutropenia. Grade 3 non-hematologic toxicities were diarrhea, vomiting, hyperglycemia, and hypernatremia (1 patient each). Other toxicities were non-cumulative grade 1-2 gastrointestinal events (8 patients with diarrhea, 3 dysgueusia, 2 vomiting, 1 nausea, 1 anorexia, 1 abdominal pain), hyperglycemia (7 patients), skin rash (3 patients), asymptomatic coagulation factor VII decrease (2 patients), and direct bilirubin increase (1 patient). Dose proportional plasma concentrations were observed and trough concentrations > 500 nM occurred regularly from 80 mg/day. Additional patients are currently being treated at 80 mg QD or with various discontinuous schedules at 120 mg (5 days on/2 days off, 2 weeks on/1 week off, 1 week on/2 weeks off) to determine the recommended regimen. Clinically relevant activity was reported in 6 patients treated from 40 to 120 mg, including one CR (120 mg, 17+ weeks [wks]) and 1 PR (80 mg, 28 wks), both in DLBCL patients failing 3-4 prior therapy lines, and both with clinical benefit. Four other patients (two with DLBCL, one follicular, and one lymphoplasmacytic lymphoma) had minor tumor shrinkage with clinical benefit (40 mg, 36+ wks; 80 mg, 14 wks; 120 mg 15 wks; 120 mg, 17 wks). Conclusions: OTX015 single agent exhibits clinically significant activity against resistant DLBCL with two responses and two minor tumor shrinkages among nine evaluable patients treated at doses ≥ 80 mg. Centralized pathology review including immunohistochemistry profiling is being performed retrospectively, and will be prospective in a DLBCL expansion cohort. Updated data including recommended regimen and correlations between clinical activity and biomarkers will be presented. Disclosures Thieblemont: Oncoethix SA: Research Funding. Stathis:Oncoethix SA: Research Funding. Inghirami:Oncoethix SA: Research Funding. Karlin:Oncoethix SA: Research Funding. Morschhauser:Oncoethix SA: Research Funding. Gleeson:Oncoethix SA: Research Funding. Broussais:Oncoethix SA: Research Funding. Amorim:Oncoethix SA: Research Funding. Salles:Oncoethix SA: Research Funding. Facon:Oncoethix SA: Research Funding. Cunningham:Oncoethix SA: Research Funding. Vey:Oncoethix SA: Research Funding. Bourdel:Oncoethix SA: Employee of study CRO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Zucca:Oncoethix SA: Research Funding.

Background: Lymphomas are still incurable in many patients and novel active compounds are actively being sought. We previously reported single agent activity of the BET bromodomain OTX015 in lymphoma cell lines and in vivo (AACR 2012; ICML 2013). Here, we report a study of the mechanism of action of OTX015 and its activity in combination with other anti-cancer compounds. Methods: Cell lines: 22 diffuse large B-cell lymphoma (DLBCL), 4 mantle cell lymphomas, 3 multiple myelomas, 3 splenic marginal zone lymphoma and 1 prolymphocytic leukemia. Anti-proliferative of OTX015 (OncoEthix SA, Switzerland) was assessed by MTT and its cytotoxic activity by Annexin V staining and gene expression profiling (GEP) with Illumina HumanHT-12 Expression BeadChips. Data mining was done with LIMMA, GSEA, Metacore. Synergy was assessed in cells (2-5 cell lines) exposed for 72 h to increasing doses of OTX015 alone or in combination with increasing doses of other agents. MTT assays were performed and Chou-Talalay combination index (CI) calculated. Results: OTX015 (500 nM, 72h) showed cytostatic activity in 29/33 (88%) cell lines and apoptosis in 3/22 (14%). Mutations in genes coding for MYD88 and components of BCR (P=0.027), and ABC signaling phenotypes (P=0.008) were significantly associated with apoptosis induction. We performed GEP on 2 cell lines (SU-DHL-6, SU-DHL-2), treated with DMSO or OTX015 (500 nM) for 1, 2, 4, 8 or 12 hours. Most upregulated genes were histones. MYC target genes were highly significantly enriched among all OTX015 regulated transcripts and MYC was the most frequently downregulated gene. OTX015 also downregulated MYD88, IRAK1, TLR6, IL6, STAT3, and TNFRSF17, members of the NFKB, TLR and JAK/STAT pathways. NFKB target genes (IRF4, TNFAIP3 and BIRC3) were also downregulated (PCR). Immunoblotting and immunohistochemistry showed a reduction of transcriptionally active pSTAT3 in 2 ABC cell lines, and a reduction in nuclear localization of p50 (NFKB1), indicating an inhibitory effect of OTX015 on the canonical NFKB pathway. Finally, IL10 and IL4 production was reduced after 24 hours OTX015 treatment. Synergy was observed with everolimus (median CI=0.1; range 0.1-0.2), ibrutinib in ABC-DLBCL (CI=0.04; 0.02-0.1), idelalisib (CAL101) (CI=0.5; 0.04-2.4), vorinostat (CI=0.5; 0.3-0.6), rituximab (CI=0.5; 0.4-0.5), decitabine (CI=0.6; 0.6-0.7), lenalidomide (CI=0.7; 0.6-0.7), and all-trans retinoic acid (CI=0.4; .1-1.6). Additive effects were observed for combinations with romidepsin (CI=1.08; 1-1.22), bendamustine (CI=0.92; 0.83-1.1), and doxorubicin (CI=0.83; 0.71-0.96). Conclusions: OTX015 is a promising candidate for targeted combination therapies. A phase I study (NCT01713582) in patients with hematological neoplasias is underway and together with our preclinical data, may support further clinical investigations. Citation Format: Eugenio Gaudio, Elena Bernasconi, Ivo Kwee, Michela Boi, Paola Bonetti, Chiara Tarantelli, Andrea Rinaldi, Monica Testoni, Maurilio Ponzoni, Anastasios Stathis, Georg Stüssi, Eugenia Riveiro, Patrice Herait, Emanuele Zucca, Francesco Bertoni. The BET Bromodomain inhibitor OTX015 targets the NFKB, TLR and JAK/STAT pathways and shows pre-clinical activity as single agent and in combination in mature B-cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5528. doi:10.1158/1538-7445.AM2014-5528

Background: The human BET family bromodomains, including BRD2-4 and BRDT proteins, has become a druggable target for the development of specific gene transcription inhibitors. Here, we report the preclinical anti-leukemic activity of OTX015 (OncoEthix SA, Switzerland), a novel pan-BET-BRD inhibitor, as single agent and in combination with different drugs. Material and Methods: Eight established human cell lines from acute and chronic myeloid leukemia (AML, i.e. HL-60 and U-937; CML, i.e. K-562 and NALM-1) and acute lymphoblastic leukemia (ALL, i.e. Jurkat, CCRF-CEM, MOLT-3 and -4) were treated by increasing doses of OTX015. The growth inhibition 50% (GI50) values of OTX015 were evaluated by MTT the assay at 72 h. Protein levels were analyzed by Western blot using commercial antibodies. RNA was extracted using the Qiagen RNAEasy and RT-PCR was performed using Fast SYBR Green on a StepOnePlus Real-Time PCR System. Combination effects were evaluated in HL60, U937, Jurkat and K562 cell lines; OTX015 was administered with daunorubicin, azacytidine, dexamethasone, cytarabine or methotrexate. The 48-h combination index (CI) was determined by median effect plot analysis using CalcuSyn software expressed as the median and range of CI values among cell lines (Chou & Talalay analysis). CI1: antagonism. Results: Firstly, the anti-proliferative effect of OTX015 was assessed. In HL60, U937 and Jurkat cells, GI50s values were between 230 and 384 nM, while ≥ 1,000 nM for the remaining cell lines. Both OTX015-sensitive and -resistant cells showed similar basal expression levels of BRD2-4, c-MYC, BCL-2, p21 and Cyclin D1 proteins. In sensitive cell lines, OTX015 caused cell cycle arrest in G1 in a time-dependent manner without induction of apoptosis. Likewise, c-MYC mRNA and protein levels were down-regulated after 6-h and up to 72-h treatments with 500 nM OTX015. In Jurkat cells, OTX015 induced up-regulation of BRD2 and 4 mRNA without modifying protein levels. On the other hand, although the OTX015-resistant cell line K562 showed decreased levels of c-MYC mRNA after 4-h and 24-h exposure, protein levels were constant even after 72 h of treatment. Synergy was observed with daunorubicin in OTX015-sensitive and -resistant cell lines (CI=0.8;0.4-0.9). The combination of OTX015 with azacytine or cytarabine was synergistic (CI=0.6;0.6-0.7 and CI=0.8;0.4-0.9, respectively) despite primary resistance of Jurkat and K562 lines to either of the individual drugs. Methotrexate, dexamethasone and panobinostat exerted synergistic effects with OTX015 in 3 out of 4 cell lines (CI=0.5;0.2-0.9; CI=0.3;0.1-0.7 and CI=0.6;0.5-0.7). Conclusion: Our findings indicate that OTX015 enhances in vitro anti-proliferative effects of standard antileukemic agents supporting combination therapy as an important aspect of the clinical development plan. Citation Format: Lucile Astorgues-Xerri, Ramiro Vázquez, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Eric Raymond, María E. Riveiro. In vitro evaluation of OTX015, a novel pan-BET-bromodomain (BET-BRD) inhibitor, as single agent and in combination with standard chemotherapy drugs in human leukemic cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5529. doi:10.1158/1538-7445.AM2014-5529

Background: OTX015 is a novel inhibitor of the bromodomain and extra-terminal (BET) protein family. This synthetic oral small molecule targets the BET transcriptional co-activator proteins BRD2/3/4, which are potential cancer targets particularly in hematologic malignancies. A phase Ib study with OTX015 administered to patients with hematologic malignancies was performed including a pharmacokinetic (PK) investigation. The PK objectives of this study were to determine the PK profile and perform a PK/PD modeling of oral OTX015 using a population approach. Materials and Methods: A multicenter, dose escalation study is underway in cohorts of 3 to 6 patients with acute leukemia and other hematologic malignancies. Patients received oral OTX015 at a starting dose of 10 mg once daily (QD). PK blood samples from 7 time points were collected over 24 h post-administration (complete PK) on Day 1 for leukemia patients and 4 blood samples over 8 h post-administration were collected for patients with other malignancies (limited sampling PK). Plasma concentrations of OTX015 were measured using validated Ultra Performance Liquid Chromatography with tandem Mass Spectrometry detection (UPLC-MS/MS) with a concentration range 1 - 250 ng/mL. Analyses and population PK (PPK) modeling were performed with the nonlinear mixed effect modeling software program Monolix version 4.2. Results: From January 2013 to January 2014, 36 patients were treated at four dose levels (10, 20, 40, 80 mg) QD and 40 mg BID. 302 plasma concentrations (289 + 13 BLQ) were analyzed. A 1-compartment open model adequately described the total OTX015 time-concentration curve. The PPK parameters obtained for the structural model were: Ka (absorption constant) = 1.12 h-1; V (distribution volume) = 68.6 L and CL (clearance) = 6.65 L/h with a relative standard error of 27%, 9% and 10% respectively. AUC values for all patients increased dose-proportionally (R²= 0.995). The absorption phase was linear and Tmax was between 1 and 4 hours. Mean elimination half-life of OTX015 for all patients was 7.16 h. The main covariate effects in PPK modeling were body weight (BW) which influenced CL, V. No significant gender influence on PK parameters was observed. Conclusions: The PK of OTX015 is best described by a one-compartment model. BW influenced significantly PK parameters of OTX015. AUC-dose proportionality was observed. Evaluation of glucuronidated metabolite concentrations is ongoing and will contribute to understanding the pathways involved in OTX015 metabolism. PK/PD modeling will be performed to describe toxicity and efficacy (6 experienced clinically meaningful activities) in terms of OTX015 PK. A comparison between QD and BID schemes will be performed in order to verify PK profile of OTX015 for each administration. Citation Format: Elodie Odore, Keyvan Rezai, Eugenia Riveiro, Fabrice Bourdel, Patrice Herait, Esteban Cvitkovic, Herve Dombret, Francois Lokiec. A phase I pharmacokinetic study of OTX015 for the treatment of patients with hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-231. doi:10.1158/1538-7445.AM2014-LB-231

Background: Various inhibitors targeting the activity of BET proteins have been recently developed and have shown potent anti-proliferative effects in several tumors, including NSCLC. The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes results in the chimeric oncogene EML4-ALK, identified as a distinct entity of NSCLC patients that enables effective ALK-targeted therapy. However, most of these patients invariably acquire resistance within few months. Herein, we report preclinical findings obtained with a novel oral pan-BET-BRD inhibitor, OTX015, in a panel of NSCLC cell lines, some of which bear the fusion protein EML4-ALK. Materials and Methods: Five established NSCLC cell lines (i.e. H2228, H3122, A549, HOP62 and HOP92) were exposed to increasing concentrations of OTX015 (OncoEthix SA, Switzerland). In each case, the effect on cell viability was determined by the MTT assay after 72 h of exposure. GI50 values, representing 50% growth inhibition concentration, were calculated using GraphPad Prism 5.0 software. Protein levels were determined by Western blot using commercial antibodies. RNA was extracted with the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. Results: OTX015 displayed anti-proliferative activity in EML4-ALK-positive H2228 and H3122 cells after a 72-h treatment, with GI50 values of 629 and 627nM, respectively. The corresponding GI50 values for the benchmark compound, JQ-1, were 2161 and 1265 nM. Interestingly, OTX015 was also active in the EML4-ALK-negative A549 cell line, with a GI50 of 432 nM. BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1 were detected at protein and mRNA levels in all cell lines. Both OTX015-sensitive and -resistant cells exhibited similar basal expression levels for the aforementioned proteins. EML4-ALK variants 1 and 3 were identified in H3122 and H2228 cells, respectively. Assessment of the signaling pathways involved in the anti-proliferative activity of OTX015 in these cells showed a transient up-regulation of STAT3, followed by a down-regulation after 24 h and up to 72 h of exposure. These results indicate that the key downstream effector of OTX015 is STAT3, frequently found to be overexpressed in crizotinib-resistant cell lines. Interestingly, c-MYC protein and mRNA levels were not altered by OTX015. EML4-ALK-positive H3122 cells showed down-regulation of n-MYC mRNA levels after OTX015 treatment. Conclusion: Our results indicate that NSCLC cell lines with genomic ALK alterations are sensitive to BET-BRD inhibition by OTX015, suggesting its potential clinical use as anticancer agent in EML4-ALK positive NSCLC patients. Citation Format: Ramiro Vázquez, Lucile Astorgues-Xerri, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Michela Boi, Giorgio Inghirami, Maurizio D'Incalci, María E. Riveiro, Eric Raymond. OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5530. doi:10.1158/1538-7445.AM2014-5530

Aim: The bromodomain and extraterminal (BET) subfamily of human bromodomain (BRD) proteins associates with acetylated chromatin and plays a key role in the epigenetic control of transcriptional activation, notably of genes with super-enhancers, such as the MYC oncogene. OTX015, a potent small molecule inhibitor of BRD2/3/4 (Noel et al, EORTC-NCI-AACR 2013), inhibits proliferation of a wide range of hematologic malignancies (HMs) in vitro (Bonetti et al, EORTC-NCI-AACR 2012; Boi et al, EORTC-NCI-AACR 2013; Braun et al, ASH 2013). This phase I study, designed to determine the recommended dose and pharmacokinetics of oral OTX015 as a single agent, is the first reported clinical study evaluating the effects of BRD inhibition in patients (pts) with HMs. Methods: Two independent cohorts of pts having failed all standard therapies were given ascending oral doses of OTX015 in a conventional 3+3 design, with acute leukemias (AL) treated 14 days on/7 days off and other HMs (OHM) treated continuously in 21-day cycles. OTX015 was given once daily (QD), then twice daily (BID). Results: From Jan to Dec 2013, 16 pts with AL (14 AML, 2 ALL) and 17 with OHM (6 DLBCL, 5 other lymphomas, 6 multiple myelomas) were enrolled over 4 dose levels, 10, 20, 40 and 80 mg QD. Exposure increased dose-proportionally. Plasma trough concentrations at 80 mg QD and 12h concentrations at 40 mg QD were ≥ IC50 values in vitro (250 nM), justifying the shift to a BID schedule. The 40 mg BID cohort is ongoing. Pts have a median age of 70 years (range 32-83) and median of 2 (1-8) prior therapies; 10 of 16 AL pts had AML secondary to pre-existing conditions or chemotherapy. No dose limiting toxicity was observed up to 80 mg QD/40 mg BID. Adverse events (AEs) were mainly grade (G) 1-2 hematologic and gastrointestinal events and diabetes aggravation. G 3-4 AEs were reversible thrombocytopenia in 3 pts with OHM (40 and 80 mg), and neutropenia, diarrhea, and elevated transaminases in 1 pt each. No cumulative toxicity was observed. Nine pts received >3 (range 4-7) cycles without or with minor interruptions. Among 28 pts evaluable for response, 6 had clinically meaningful activity, with 4 of 6 treated at 80 mg. Four pts with refractory/relapsed secondary or post-treatment AML achieved significant peripheral and bone marrow blast decrease or clearance, including 1 complete remission (CR) and 1 CR with incomplete recovery. Among OHM, 1 DLBCL had a partial response (PR) and 1 lymphoplasmacytic lymphoma had metabolic PR on cycle 2 PET-scan. Treatment of 5 of 6 responding pts is ongoing. Responses occurred in pts with various clinical, cytogenetic and molecular profiles. Conclusion: OTX015 is the first BRD inhibitor demonstrating clinical activity. Maximum tolerated dose was not reached at 80 mg QD or 40 mg BID; dose escalation is ongoing with the BID schedule and further schedule optimization. Updated results will be presented. Citation Format: Patrice E. Herait, Celine Berthon, Catherine Thieblemont, Emmanuel Raffoux, Valeria Magarotto, Anastasios Stathis, Xavier Thomas, Xavier Leleu, Carlos Gomez-Roca, Elodie Odore, Christophe Roumier, Fabrice Bourdel, Bruno Quesnel, Emanuele Zucca, Mauricette Michallet, Christian Recher, Esteban Cvitkovic, Keyvan Rezai, Claude Preudhomme, Thierry Facon, Antonio Palumbo, Herve Dombret. BET-bromodomain inhibitor OTX015 shows clinically meaningful activity at nontoxic doses: interim results of an ongoing phase I trial in hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT231. doi:10.1158/1538-7445.AM2014-CT231

Background Bromodomain and extra-terminal (BET) proteins, including the ubiquitous BRD2/3/4 proteins, are epigenetic readers implicated in c-MYC transcription, cellular proliferation, cell-cycle progression, RNA elongation and DNA damage response. Using shRNA screening and BRD inhibitors, BRD4 has been established as a promising therapeutic target in acute leukemia (Zuber, Nature 2011). In the present study, we investigated the in vitro anti-leukemic effects of the small-molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland). Methods Expression of BRD2/3/4 and c-MYC was assessed by RQ-PCR in 5 myeloid (HL60, KG1, KG1a, K562, NOMO1) and 4 lymphoid (Jurkat, RS4-11, BV173, TOM1) leukemia cell lines and by Western blotting (WB) using commercial antibodies in the HL60, K562, Jurkat and RS4-11 lines. Nineteen AML and ten ALL patient banked leukemic cells were assayed by RQ-PCR only. Cell viability and IC50 values were assessed in cell lines by MTT assays after exposure to OTX015 (0.1nM-10µM) for 72h. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Induction of apoptosis was evaluated in cell lines and patient cells by outer membrane phosphatidylserine exposure and PI incorporation at 72 hours with increasing doses of OTX015 (25nM-500nM). Caspase-3 activation and mitochondrial cytochrome c release were studied by immunofluorescence (IF). Maturation was assessed by morphological studies after MGG staining and detection of CD11b by FACS analysis. Modulation of BRD2/3/4 proteins was investigated by WB. Results OTX015 IC50 values were in the submicromolar range for KG1 and the MLL-driven NOMO1 cell lines (198.3 and 229.1nM, respectively), while K562 was the most resistant myeloid line, with an IC50 of 11.3µM. In contrast, in lymphoid cell lines tested, IC50 values ranged from 34.2 to 249.7nM, with the MLL-driven cell line RS4-11 being the most sensitive. Cell cycle arrest in subG1/G1 to S transition was observed in 8/9 cell lines and was most pronounced in RS4-11 and BV173. Significant apoptosis (up to 88% Annexin V positive cells) was only observed in KG1a and NOMO1 among myeloid cell lines, while OTX015 induced apoptosis in all lymphoid cell lines tested, ranging from 57% in RS4-11 to 90% in the BCR-ABL+ TOM1 cells. Similarly, OTX015 triggered caspase-dependent cell death, as NOMO1 and RS4-11 displayed significant caspase-3 activation and cytochrome c release, when compared to the resistant K562 cell line. Seven primary patient fresh samples (5 AML, 2ALL) were also analyzed. Ex vivo treatment induced apoptosis ranged from 35% to 87% in 6/7 patients. Exposure to OTX015 at 500nM for 7 days induced maturation in 51% and 65% of HL60 cells as detected by CD11b expression and morphology, respectively. Baseline expression of BRD2/3/4 varied among cell lines or patient samples, lower BRD2/3/4 expression levels were observed in the BCR-ABL+ K562 and BV173 cell lines, as well as in the 4 BCR-ABL+ ALL samples analyzed. Upon OTX015 exposure, down-regulation of the BRD4 target gene c-MYC was observed in all cell lines, without clear correlation with the proliferation inhibition rate and/or the intensity of induced apoptosis while no consistent BRD2/3/4 mRNAs down-regulation was seen. Interestingly, BRD2 protein was down-regulated in HL60, Jurkat and RS4-11 cell lines, but not in the K562 cell line. Conclusion OTX015 affects cell viability, induces cell cycle arrest in G1/S phase, and is able to induce significant apoptosis in leukemic cell lines and fresh AML and ALL samples at submicromolar drug concentrations. These concentrations were achieved in the serum of healthy volunteers after safe administration of the drug. With such characteristics, OTX015 appears to be an attractive anti-leukemic therapy, currently under early evaluation in a Phase Ib dose-escalation trial conducted in relapsed/refractory AML/ALL patients. Disclosures: Riveiro: Oncology Therapeutic Development: Employment. Herait:Oncoethix: Employment. Dombret:Oncoethix: Research Funding.

Epigenome deregulation in cancer cells affects transcription of oncogenes and tumor suppressor genes. BET Bromodomain proteins recognize chromatin modifications and act as epigenetic readers contributing to gene transcription. BET Bromodomain inhibitors showed promising pre-clinical activity in hematological and solid tumors and are currently in phase I studies. The mechanism of action and relevant affected genes are not fully characterized and there are no established response predictors. We have shown activity of BET Bromodomain OTX015 in lymphoma cell lines (ASH 2012; ICML 2013). This study aimed at elucidating pathways and genes affecting response/resistance to BET Bromodomain inhibitors in lymphomas. Methods Baseline gene expression profiles (GEP) were obtained in 38 cell lines [22 diffuse large B-cell lymphoma (DLBCL), 8 anaplastic large T-cell lymphoma, 4 mantle cell lymphoma, 3 splenic marginal zone lymphoma, 1 chronic lymphocytic leukemia] with Illumina HumanHT-12 v4 Expression BeadChip. Genetic and biologic information were collected from literature. GEP/IC50 correlation (ASH 2012; ICML 2013) was assessed by Pearson correlation. Associations in two-way tables were tested for statistical significance using either chi-square or Fisher exact test, as appropriate. Differential expression analysis was performed using LIMMA, followed by multiple test correction using the BH method. Enrichment of functionally-related genes was evaluated by GSEA. For combination studies, 3 germinal center B-cell (GCB) and 2 activated B-cell (ABC) DLBCL were exposed to increasing doses of OTX015 alone or in combination with increasing doses of targeted agents for 72 hours, followed by MTT assay. Synergy was assessed by Chou-Talalay combination index (CI) with Synergy R package. Results Transcripts associated with resistance to OTX015 were significantly enriched of genes involved in cell cycle regulation, DNA repair, chromatin structure, early B-cell development, E2F/E2F2 target genes, IL6-dependent genes, and mRNA processing. Conversely, transcripts associated with OTX015 sensitivity were enriched of hypoxia-regulated genes, interferon target genes, STAT3 targets, and involved in glucose metabolism. Genes associated with OTX015 sensitivity included LDHA, PGK1 (glucose metabolism) and VEGFA (hypoxia), while BCL2L1/BCLXL, BIRC5/survivin (anti-apoptosis), ERCC1 (DNA repair), TAF1A and BRD7 (transcription regulation) were correlated with reduced sensitivity. GEP identified 50 transcripts differentially expressed, including IL6, HCK, SGK1, MARCH1 and TRAFD1, between cells undergoing or not apoptosis after OTX015 exposure. GSEA showed significant enrichment of genes involved in IL-10 signaling pathway. While there was no association between response to OTX015 Conclusions Our study identified genetic mechanisms contributing to the response to BET Bromodomain inhibitors and promising combination schemes, such as OTX015/everolimus, to be further investigated. Disclosures: Stathis: Oncoethix: NCT01713582 PI Other. Herait:Oncoethix: Membership on an entity’s Board of Directors or advisory committees. Noel:Oncoethix: Membership on an entity’s Board of Directors or advisory committees. Inghirami:Oncoethix: Research Funding. Bertoni:Oncoethix: Research Funding.

Background: Gal-1 is a lectin with multiple biological functions including tumor progression, migration, and angiogenesis. OTX008, a small molecule downregulating gal-1 protein level, displays direct antiproliferative and anti-invasive effects in human cancer cells. Objectives: Determine the maximum tolerated dose (MTD) of single agent OTX008 using a subcutaneous (SC) daily dosing based on dose-limiting toxicities (DLTs). Secondary objectives were safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity. Patients and Methods: Patients (pts) with solid tumors having failed standard therapies, and given a written consent were enrolled. OTX008 was delivered as daily SC injection. In case of DLT, treatment was interrupted until recovery and resumed at the dose level (DL) below. Dose was escalated according to a 3+3 design. The MTD was defined as the highest DL, in which ≤1/6 pt experiences a DLT. Results: 22 pts (50% with colorectal carcinoma) were treated from March 2012 to March 2013. Six were enrolled at DL1 (65mg-flat dose) without DLT. Among the 7 pts enrolled at DL2 (120-130mg), 2 experienced DLT. DL2 was therefore deemed to exceed the MTD and 9 additional pts were enrolled at DL1. No DLT was observed among the 15 patients treated at DL1. The most frequent related adverse event (AE) was G1-2 injection site reaction in 20 patients. Two pts experienced skin ulcerations, resulting in subcutaneous abscess and treatment discontinuation in one and consent withdrawal in another. Transient and fully reversible neurological AEs were observed in 12 pts (55%), and were more frequent (100% vs 33%) and more severe (G3 in 29% vs 0%) at DL2 compared to DL1 respectively. G 1-2 tremor was observed in 8 pts, perioral paresthesia in 5, dizziness in 4 and myoclonia in 2. Two patients experienced G3 ataxia (DLT), and one patient with past history of post-traumatic epilepsy experienced G3 seizure. Gastrointestinal (GI) AEs were reported by 12 pts (55%); G3 vomiting/abdominal pain and G3 diarrhea were reported in 1 pt each; 12 patients developed asymptomatic abnormal lab values (10 hypomagnesemia and 8 transient elevation of CPK without rhabdomyolysis or cardiac ischemia). OTX008 plasma concentrations were >1µM over 12h after administration. PK of OTX008 is best described by a two compartment open model and showed less than proportional exposure increase, possibly due to wide inter-patient variability. The main covariate effect was related to body weight. Plasma gal-1 levels decreased proportionally with increasing OTX008 plasma concentrations. Serial tumor biopsies in 6 patients did not conclude on a PD effect. No objective response was reported. Conclusions: OTX008 recommended flat daily sc dose is 65mg, which achieves significant systemic plasma concentrations based on in vitro models. Weight adapted dosing may optimize systemic exposure. Local tolerance of SC injection is poor.Reversible ataxia was a dose-limiting toxicity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A72. Citation Format: Jean-Pierre Delord, Ahmad Awada, Eric Raymond, François Lokiec, Patrice Herait, Keyvan Rezai, Nicolas Lachaux, Gabriel A. Rabinovich, Carlos Gomez-Roca, Philippe Aftimos, Sandrine Faivre, Juan Carlos Stupirski. A first-in-man Phase I study of the galectin-1 (gal-1) inhibitor OTX008 given subcutaneously as a single agent to patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A72.

Background: Galectin-1, a multifunctional lectin, modulates cancer cell proliferation and tumor angiogenesis. OTX-008, a synthetic calixarene molecule, binds directly to galectin-1 and induces a conformation change in reduces galectin-1 binding to carbohydrates. In vitro pharmacodynamic studies showed that OTX008, at micromolar concentrations, inhibited agglutination, proliferation, and invasion of cultured cancer cells. Objectives of the ongoing OTX008 phase I study are: determine the recommended dose, pharmacokinetics (PK), pharmacodynamics (PD) and a PK/PD modeling of OTX008 given subcutaneously (SC) in humans. Materials and Methods: This is a multicenter, dose escalation, open label, phase I study in successive cohorts of 3 to 6 evaluable patients treated with OTX008. Nine time point blood samples were collected on D1 of cycle 1 and two blood samples, just before and 1 hour after OTX008 administration (Cmin/Cmax) were collected on D2 and D22. Urine samples were collected during the first 24 hours after D1 administration. At the first DL (65 mg OTX008 sq qd), tumor samples also were obtained from a patient with advanced, heavily pre-treated cutaneous angiosarcoma of the scalp at baseline and at D22. Plasma, urine and tumor concentrations of OTX008 were measured using a validated Ultra Performance Liquid Chromatography with tandem Mass Spectrometry detection (UPLC-MS/MS) with range of 5 - 250 ng/mL. Pharmacokinetic analyses were carried out using the nonlinear mixed effect modeling software program Monolix version 4.1. Results: PK profiles of 7 patients treated at DL1 (65 mg) are reported. A 2-compartment open model adequately described the total OTX008 time-concentration curve with linear elimination. Following D1 administration, mean + SD Cmax was 4666 + 2012 ng/ml; and Tmax ranged from 0.5 h to 2 h; mean + SD AUC was 48.9 + 36.8 mg.h/l and mean + SD T1/2 was T1/2=5.5 + 0.9 h. Prior to D2 dosing, all patients had OTX008 plasma concentrations ≥1 μM. No accumulation was observed, steady state was reached around D22 after drug administration; 8-10 % of dose was recovered in urine during the first 24 hours. In one patient assessed, the intra-tumor concentration of OTX008 at D22 was 0.002 nM/mg of tumor. DL2 130mg sq qd ongoing (ongoing) and 65 mg bid schedule (planned) will be assessed and also presented. Conclusions: Our DL1 results show that OTX008 is rapidly absorbed and distributed after single SC administration. The PK of OTX008 is best described by a two compartment open model. The influence of BMI will be studied with further DL cohorts and larger number of patients. Urinary excretion seems to be the major route of unchanged drug elimination. High plasma concentrations, close to the active dose in vitro were achieved from DL1. Citation Format: Keyvan Rezai, Sylvere Durand, Nicolas Lachaux, Eric Raymond, Patrice Herait, Francois Lokiec. OTX008 pharmacokinetics (PK) during the first-in-man phase I study in patients with advanced solid tumors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 33. doi:10.1158/1538-7445.AM2013-33

Summary Background Irinotecan (CPT-11) is a novel water-soluble camptothecin derivative selected for clinical testing based on its good in vitro and in vivo activity in various experimental systems, including pleiotropic drug-resistant tumors. Its mechanism of action appears mediated through topoiso-merase I inhibition. The purpose of this study was to describe CPT-11 and active metabolite SN-38 population pharmacokinetics, examine patient characteristics that may influence pharmacokinetics, and to investigate pharmacokinetic-phar-macodynamic relationships that may prove useful in the future clinical management of this drug. Patients and methods As part of 3 Phase I studies including 235 patients, pharmacokinetics of CPT-11 and metabolite SN-38 were determined in 107 patients. CPT-11 was administered as a 30-min i.v. infusion according to 3 different schedules: daily for 3 consecutive days every 3 weeks, weekly for 3 weeks, and once every 3 weeks. Patients characteristics were the following: median age 53 years; men, 45 women; 105 Caucasians, 2 blacks; performance status was 0–1 in 96 patients; tumor sites were predominantly colon, rectum, head and neck, lung, ovary and breast; with the exception of 6 patients, all had been previously treated with surgery, chemotherapy and/or radiotherapy. CPT-11 and metabolite SN-38 were simultaneously determined by HPLC using fluorescence detection. Pharmacokinetic parameters were determined using model-independent and model-dependent analyses. Results 168 pharmacokinetic data sets were obtained in 107 patients (97 first courses, 43 second courses, 23 third courses, 4 fourth courses, and 1 fifth course). Rebound concentrations of CPT-11 were frequently observed at about 0.5 to 1 h following the end of the i.v. infusion, which is suggestive of enterohepatic recycling of the drug. Model-independent analysis yielded the following mean population pharmacokinetic parameters for CPT-11: a terminal half-life of 10.8 h, a mean residence time (MRT) of 10.7 h, a volume of distribution at stedy state (Vdss) of 150 L/m2, and a total body clearance of 14.3 L/m2/h. Model-dependent analysis disclosed a CPT-11 plasma disposition as either biphasic or tri-phasic with a mean terminal half-life of 12.0 h. The volume of distribution Vdss (150 L/m2) and total body clearance (14.8 L/m2/h) yielded almost identical values to the above model-independent analysis. The active metabolite SN-38 presented rebound concentrations in many courses at about 1 h following the end of the i.v. infusion which is suggestive of enterohepatic recycling. The mean time at which SN-38 maximum concentrations was reached was at 1 h since the beginning of the 0.5 h infusion (i.e., 0.5 h post i.v.). SN-38 plasma decay followed closely that of the parent compound with a mean apparent terminal half-life of 10.6 h. Mean 24 h CPT-11 urinary excretion represented 16.7% of the administered dose, whereas metabolite SN-38 recovery in urine was minimal (0.23% of the CPT-11 dose). The number of CPT-11 treatments did not influence pharmacokinetic parameters of either the parent compound or metabolite SN-38. Although CPT-11 pharmacokinetics presented an important interpatient variability, both CPT-11 maximum concentrations (Cmax) and the CPT-11 area under the plasma concentration versus time curves (AUC) increased proportionally and linearly with dosage (Cmax, r=0.78, p Patient physio-pathological characteristics were examined as possible determinant of pharmacokinetics. No detectable relationship was observed between CL or the metabolic ratio (% SN-38 AUC/CPT-11 AUC), with the following physio-pathological factors: age, sex, height, weight, body surface, tumor type, or renal function. However, with regard to hepatic function, significant correlations (negative) were observed with CPT-11 CL and some hepatic function markers, e.g., bilirubinemia and gamma-glutamyl transpeptidase. Also of interest, a significant positive correlation between the metabolic ratio and some liver function parameters were observed, e.g., bilirubinemia, aspartate transferase (AST), and alanine transferase (ALT). For the pharmacokinetic-pharmacodynamic studies, CPT-11 AUC correlated significantly with the percent decrease of either the white blood cells or the neutrophils. CPT-11 AUC also correlated significantly with the intensity of diarrhea, and the intensity of nausea and vomiting. CPT-11 CL correlated negatively with the intensity of the principal toxicities observed. Metabolite SN-38 AUC was also significantly correlated with the percent decrease in white blood cells and neutrophils. Significant correlations were also observed between SN-38 AUC and the intensity of diarrhea, and the intensity of nausea and vomiting. The metabolic ratio did not correlate with any of the principal toxicities encountered in these clinical studies. With regard to antitumor responses, although the optimal schedule and dose were not obviously defined at the beginning of these phase I trials, 17 tumor responses were nevertheless observed (2 complete, 9 partial, 6 minor). The observation that most of these responses were obtained at the highest doses administered is highly suggestive of a dose-response relationship with this drug. Conclusions These data indicate that CPT-11 population pharmacokinetics is linear within the large dose range investigated, that the number of treatments do not influence pharmacokinetics, that liver function affects CPT-11 clearance. Also of interest, the intensity of the major toxicities encountered with this drug (e.g., leukoneutropenia, diarrhea, nausea and vomiting) correlated with the exposure (AUC) to CPT-11 and metabolite SN-38. A dose-effect relationship was also noted for anticancer activity since most tumor responses were observed at the highest doses administered. These pharmacological data are of importance for the conduct of future clinical studies with this active drug.

Background. Galectin-1 is a multifunctional protein involved in various aspects of tumorigenesis and has been described as a promising cancer target. It has been previously shown that galectin-1 has high sensitivity to oxidative inactivation. During oxidation, galectin-1 may forms disulfide bridges resulting in profound conformational changes, which prevent galectin-1 dimerization and ligand recognition. OTX-008 is a non-peptide chemical antagonist designed to bind galectin-1. We previously showed that OTX-008 displays direct antiproliferative effects and inhibits galectin-1 expression in cancer cell lines (AACR 2011, Abstract n°685). The aim of the present study was to further the understanding on the molecular mechanism implicated on galectin-1 downregulation mediated by OTX-008 in cancer cells. Material and Methods. Proliferative SQ20B cancer cells were exposed to 3µM OTX-008 during 48h with or without i) 10µM N-acetylcysteine (an antioxidant agent), ii) 1mM Tempol (an antioxidant agent), iii) 10µM co-enzyme Q10 (a naturally occurring antioxidant agent), iv) 55µM α-mercaptoethanol (a strong reducing agent), or v) 10nM bortezomib (a proteasome inhibitor). Effects on galectin-1 protein levels were assessed by western blot analysis. Results. The head and neck SQ20B human cancer cell line is sensitive to OTX-008 (GI50 = 3µM). In this cell line, 48h-exposure to OTX-008 inhibits galectin-1 protein level (40% inhibition respect to control cells) without effect on galectin-1 mRNA levels. It has been described that galectin-1 oxidation represents a mechanism by which galectin-1 activity is regulated. SQ20B cells were exposed to OTX-008 for 48h with or without co-treatment with well-known antioxidant or reducing agents. Interestingly, these agents counteract the effects of OTX-008 on galectin-1 expression (galectin-1 protein levels by densitometry analysis: control SQ20B 100%, OTX-008 60%, N-acetylcystein+OTX-008 110%, Tempol+OTX-008 120%, co-enzyme Q10 +OTX-008 140% and α-mercaptoethanol+OTX-008 120%), suggesting that oxidation plays a key role in galectin-1 down-expression by OTX-008 in SQ20B cells. We further observed that pre-treatment with bortezomib abrogated the decrease in galectin-1 protein levels by OTX-008, pointing out that oxidized galectin-1 is recognized and degradated by the proteasome system. Conclusion. Our findings show that OTX-008 mechanism of action involves galectin-1 oxidation followed by proteosomal degradation in SQ20B cells. The mechanism by which OTX-008 can enhance the oxidation of galectin-1 will require further investigations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2836. doi:1538-7445.AM2012-2836

Background. Galectin-1 binds neuropilin-1, enhances VEGFR2 signalling, and contributes to membrane anchorage of H-RAS in cancer cells. Those effects may modulate cancer cell proliferation, apoptosis, and tumor angiogenesis. OTX-008 is a non-peptide chemical antagonist designed to bind galectin-1. We previously showed that OTX-008 displays direct antiproliferative effects and inhibits galectin-1 expression in cancer cell lines (AACR 2011, Abstract n°685). In this study we focused on the antitumor and antiangiogenic effects of galectin-1 inhibition by OTX-008 or shRNA in xenograft models. Material and Methods. A2780-1A9 (1A9) ovarian cells, head and neck SQ20B and SQ-shGAL-1 (SQ20B transfected with shGalectin-1 RNA) cells were injected s.c. into female nu/nu athymic mice. When tumors became palpable, 1A9- and SQ20B- mice were treated with vehicle (PBS) or 5-10 mg/kg OTX008 i.p. q.d for 3 weeks. At the end of treatment, mice were sacrificed and tumors collected. Immunohistochemistry was performed on OCT-embedded tumor stained with H&E, galectin-1, MIB1 (Ki67), VEGFR2 or CD31. Image quantification was done with Histolab software (Microvision, France) subtracting the background. Results. OTX-008 inhibited tumor growth in both 1A9 and SQ20B xenografts after 21 days of treatment. A major decrease in the number of secondary tumors development was observed in SQ20B xenografts. A decrease in galectin-1 expression was seen in treated tumors respect to control tumors (1A9: 11.103 +/− 2.103 µm2 vs 26.103 +/− 6.103 µm2, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1926. doi:1538-7445.AM2012-1926

Background: Galectin-1 binds neuropilin-1, enhances VEGFR2 signalling, and contributes to membrane anchorage of H-RAS in cancer cells. Those effects may modulate cancer cell proliferation, apoptosis, cell cycle, and tumor angiogenesis. OTX008 is a non-peptide chemical antagonist designed to bind the amphipathic -sheet conformation of galectin-1. We previously showed that OTX008 displays direct antiproliferative effects in cancer cell lines (AACR 2011, Abstract n°685). In this study we focused on the antiproliferative and antiangiogenic effects of OTX008 in xenograft models. Material and Methods: A2780–1A9 (1A9) ovarian cells and SQ20B head and neck cells, selected for intermediate/high in vitro sensitivity to OTX008, were injected s.c. into female nu/nu athymic mice. When tumors became palpable, treatment was initiated with vehicle (PBS) or 5–10 mg/kg OTX008 i.p. q.d for 3 weeks. At the end of treatment, mice were sacrificed and tumors collected. mRNA expression was evaluated with qRT-PCR. Immunohistochemistry (IHC) was performed on OCT-embedded tumor stained with H&E, MIB1 (Ki67), VEGFR2, CD31, or galectin-1. Image quantification was done with Histolab software (Microvision, France) subtracting the background. Results: OTX008 inhibited tumor growth in both 1A9 and SQ20B xenografts. OTX008 also decreased the number of secondary tumors (subcutaneous metastases distant from the injection site) in SQ20B xenografts. 1A9 and SQ20B tumors treated with OTX008 expressed significantly lower galectin-1 protein as compared to control tumors (1A9: 11.103 +/− 2.103 μm2 vs 26.103 +/− 6.103 μm2, p Conclusion: Antiproliferative activity of OTX008 in xenograft models seems to be associated with galectin-1 expression inhibition. In addition to this direct effect, qualitative and quantitative normalization of microvessels may also play a role in the antitumor activity of OTX008. Galectin-1 and VEGFR2 expression as well as quantitative assessment of microvessels are candidate biomarkers for further clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C76.

Background. Sensitivity to targeted agents inhibiting VEGFR/PDGFR signaling, including sunitinib, may be dependent of the concomitant modulation of alternative survival pathways. The lectin galectin-1 (gal1) is overexpressed in several tumors, selectively binds neuropilin-1 and enhances VEGFR2 signaling thereby activating the MAPK and/or AKT pathways in cancer cells. Those effects may result in modulation of cancer cell proliferation, apoptosis, cell cycle, and tumour angiogenesis. Optimizing the inhibition of gal1/neuropilin-1/VEGFR-dependent signaling may be a useful approach to improve the activity of multitargeted drugs in cancer cells. The aim of the present study was to evaluate the effects of PTX-008, a novel drug that targets gal1, alone or in combination with sunitinib on several human cancer cell lines. Material and Methods. Antiproliferative effects were evaluated in human colon, breast, ovarian, head & neck, lung, prostate and HCC cancer cell lines by MTT assay. Effects of PTX-sunitinib combinations were determined by median effect plot analysis (Chou & Talalay). Results. Antiproliferative effects of sunitinib as single agent were evaluated on a panel of 20 human cancer cell lines including SQ20B, COLO205 and HT29 with IC50=2; 3 and 8µM, respectively. Antiproliferative effects of single agent PTX-008 were detected in several cell lines including head & neck SQ20B, colon HT29 and COLO205, lung HOP62 and ovarian cancer cells OVCAR3 and IGROV1 with GI50 of 3; 9; 9; 27; 3 and 27µM respectively. In contrast, head & neck HEP2, prostate DU145, breast MCF7 and SKBR3, renal CAKI1, hepatocarcinoma SK-HEP1 and colon HCC2998 lines appear to be resistant to PTX-008 (GI50>27µM). Effects of PTX-008 were slowing cell proliferation and G2/M accumulation, along with blockage of ERK and AKT survival pathways. In order to evaluate the effects of PTX-008 in combination with other multitargeted inhibitors, we used sequential and simultaneous exposure to PTX-008 with sunitinib. Combinations of PTX-008 with sunitinib in two colon cancer cell lines COLO205 and HT29 (which express low gal1, VEGFR and PDGFR mRNA), result in additive (CI=1) and synergistic (CI Conclusion. Combination of PTX-008 with sunitinib appears to be synergistic both in PTX-008-sensitive and -resistant cells, suggesting that modulation of gal1 signaling may be used to enhance the activity of sunitinib. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1548.

Rationale: BET-bromodomain (BRD) proteins play a major role in the epigenetic regulation of gene transcription, notably of genes with superenhancer promoter regions including many oncogenes, such as MYC. OTX015 is a specific BRD 2, 3 and 4 inhibitor that blocks oncogene transcription, and triggers growth inhibition and apoptosis in acute leukemia cell lines and patient cells in vitro (Braun et al. ASH Annual Meeting 2013). Based on these findings, a Phase 1 study of OTX015 was designed for patients with advanced acute leukemia. Patients & Methods: Patients with various unselected relapsed/refractory leukemia subtypes for which no standard therapy options were available were enrolled in this ongoing Phase 1 study. Patients aged < 60 years had to have failed at least two lines of therapy and those aged >60 years at least one line. At least 5% bone marrow leukemic blasts were required at study entry. OTX015 was given orally, daily for 14 days of 21-day cycles (cy). The dose was escalated from 10 to 160 mg daily (QD) according to a standard 3+3 dose-escalation design, to determine the maximum tolerated dose (MTD) or biologically optimal dose. A BID schedule was tested at dose level (DL) 4 (40 mg x 2) and a continuous schedule at 120 mg. Pharmacokinetics was studied on day 1 and residual concentrations were measured on days 2, 8 and 15. Responses were assessed on blood and bone marrow aspirations at baseline, days 8, 22 and 43. Blasts at baseline and day 8 were stored for pharmacodynamic biomarker evaluation. Cytogenetic and molecular markers were collected based on center practice. Results: From January 2013 to June 2014, 36 patients were treated over 6 dose levels: 33 with acute myeloid leukemia (AML), 2 with acute lymphoblastic leukemia and 1 with refractory anemia with excess blasts. Median age was 70 years (range 19-85), 20 patients were male, 29 patients had ECOG 0-1, and 16 AML patients had normal karyotype. Patients had a median of 2 prior therapy lines (range 1-4). The median number of OTX015 cycles administered was 2 (range 1-14+), including 9 patients with >3 cycles. Among the 28 patients evaluable for dose limiting toxicity (DLT), no DLTs were observed through DL5 (120 mg QD). The MTD was exceeded at DL6 (160 mg QD) with one patient experiencing grade 3 diarrhea and another grade 3 fatigue and anorexia. The main toxicities were non-cumulative grade 1-2 gastrointestinal events (6 patients diarrhea, 3 dysgueusia, 3 abdominal pain, 3 nausea, 1 anorexia), hyperglycemia (3 patients), coagulation factor VII decrease (6 patients) and direct bilirubin increase (3 patients) (two latter AEs asymptomatic). These toxicities were mainly observed at QD doses above 80 mg and with 40 mg BID. Dose proportional plasma concentrations were observed and trough concentrations > 500 nM (in vitro active concentrations) were regularly observed from 80 mg/day. Clinically relevant activity was reported in 5 AML patients treated at 10, 40 and 80 mg, including one sustained CR from cy 4 to cy 12 (40 mg QD) and one CR with incomplete platelet recovery (CRp) from cy 2 to cy 5 (80 mg QD). Two patients (10 mg QD, 40 mg QD) had partial blast clearance (disappearance of peripheral blasts and decrease >50% in bone marrow blast percentage) and the remaining patient (40 mg BID) had gum hypertrophy resolution. Four of these 5 patients had secondary or therapy-related AML, 4 had normal karyotype and 2 had an NPM1 gene mutation. Conclusions: OTX015 single agent exhibits antileukemic activity over a wide range of DLs and plasma concentrations in patients with advanced AML. MTD is exceeded at 160 mg QD. The safe recommended dose and schedule is close to being identified. Central extensive molecular marker analysis is being performed and will be prospectively implemented in an expansion cohort. Updated data will be presented and will include correlations between regimen, pharmacokinetics, clinical activity and molecular profile. Table Dose (Schedule) N pts evaluable Evidence of activity DLT 10 QD (14/21) 3 1 20 QD (14/21) 3 40 QD(14/21) 4 1 (CR) 80 QD(14/21) 3 2 (1 CRp) 40 BID (14/21) 6 1 120 QD (14/21) 3 120 QD (21/21) 3 160 QD (14/21) 3 Diarrhea (1) Anorexia/fatigue (1) Disclosures Dombret: Oncoethix SA: Research Funding. Preudhomme:Oncoethix SA: Research Funding. Berthon:Oncoethix SA: Research Funding. Raffoux:Oncoethix SA: Research Funding. Thomas:Oncoethix SA: Research Funding. Vey:Oncoethix SA: Research Funding. Gomez-Roca:Oncoethix SA: Research Funding. Ethell:Oncoethix SA: Research Funding. Yee:Oncoethix SA: Research Funding. Bourdel:Oncoethix SA: Employee of study CRO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Michallet:Oncoethix SA: Research Funding. Recher:Oncoethix SA: Research Funding. Roumier:Oncoethix SA: Research Funding. Quesnel:Oncoethix SA: Research Funding.

A series of 21 patients (5 refractory anemias with an excess of blasts in transformation and 16 acute leukemias) were treated with small doses of ARA-C (10 mg/sq m/12 hr for 15–21 days). Improvement was noted in 15 cases (71%) and complete remission observed in 12 (57%). Complete remission was obtained after one course of treatment in 8 cases. The fact that these patients entered remission relatively slowly and did not suffer marrow aplasia suggests that low-dose ARA-C may function in vivo as it does in vitro, i.e., by inducing differentiation of leukemic blasts.