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By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.

Background: Due to their basal stress phenotype associated to transformation, cancer cells are addicted to non-mutated, non-oncogenic proteins that do not bear such vital functions in normal cells, a phenomenon referred as Non-Oncogenic Addiction (NOA). Targeting these NOAs could therefore induce selective killing of cancer cells, opening several therapeutic opportunities. Recent data suggest that the stress-related scaffold protein AAC-11 (anti-apoptosis clone 11, also known as Api5) is critically involved in cancer cells resistance to chemotherapies, metastatic potential and escape from the immune system. Methods: We have developed inactivating peptides based on the fusion of a cell penetrating sequence and portions of the leucine-zipper domain of AAC-11, which functions as a protein-protein interaction module. These peptides induce cancer cells death, through the inhibition of protein-protein interactions between AAC-11 and its partners, while sparing normal cells. We now describe further characterization of our lead peptide, JRT39, which contains residues 377 to 379 of AAC-11 linked to the cell-penetrating peptide “penetratin”. Results: In vitro, JRT39 causes cell death in a wide spectrum of cancer cell lines with IC50 ranging from 5 µM to 30 µM depending on tumor cell type. In particular, JRT39 showed selective efficacy towards primary cells from Acute Myeloid Leukemia (AML), Acute Lymphocytic Leukemia (ALL) or Sézary syndrome (SS) patients, while sparing normal hematopoietic cells, with an IC50 of 5-15 µM. Mechanistically, JRT39 induces membranolysis of cancer cells through binding to p21-activated kinase 1 (PAK1) in AML or SS cells plasma membrane, where PAK1 is overexpressed. In addition, JRT39 exerted potent anti-tumor activity in vivo in disseminated or subcutaneous AML, APL (Acute Promyelocytic Leukemia) and SS-patient derived preclinical murine models. Preliminary pharmacokinetic studies revealed that JRT39 is stable in human serum and has a plasma half-life of ~1.5-2.5 hours after intravenous (IV) administration to dogs or cynomolgus monkeys, with concentration-time data fitting 2-compartment model. Finally, JRT39 was well tolerated at 5 and 10 mg/kg after single or repeated (daily) IV injections. Conclusions: Combined, our preclinical data confirm that interfering with AAC-11-related survival pathways is a promising novel anticancer strategy and support the development of JRT39 for the treatment of cancer. Citation Format: Louise Jeammet, Emile Adicéam, Justine Habault, Anna Kaci, Jeannig Berrou, Mélanie Dupont, Nicolas Thonnart, Ewa Pasquereau-Kotula, Anne Marie-Cardine, Armand Bensussan, Marika Pla, Hervé Dombret, Claude Gardin, Martine Bagot, Jean-Christophe Rain, Hélène Sicard, Jérôme Tiollier, Thorsten Braun, Jean-Luc Poyet. AAC-11 survival pathways as therapeutic target in cancer: AAC-11 leucine-zipper domain derived peptides exert potent antitumor effects and exhibit favorable stability, pharmacokinetic and toxicology profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 464.

Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.

Patients with FLT3-ITD mutated (FLT3-ITD+) Acute Myeloid Leukemia (AML), have frequently relapsed or refractory disease and FLT3-ITD+ inhibitors have limited efficacy. Rho kinases (ROCK) are constitutively activated by FLT3-ITD+ in AML via PI3 kinase and Rho GTPase. Upon activation by ROCK, LIM kinases (LIMK) inactivate cofilin by phosphorylation which affects cytoskeleton dynamics, cell growth and apoptosis. LIMK inhibition leads to cofilin activation via dephosphorylation and activated cofilin localizes to mitochondria inducing apoptosis. Thus, we investigated the therapeutic potential of the LIMK1/2 inhibitor CEL_Amide (LIMKi) in FLT3-ITD+ AML. Expression of LIMK1/2 in FLT3-ITD+ cell lines MOLM-13 and MV-4-11 cells could be detected by RT-qPCR and at the protein level. IC50 after LIMKi monotherapy was 440 nM in MOLM-13 cells and 420 nM in MV4-11 cells. Treatment with LIMKi decreased LIMK1 protein levels and repression of inactivating phosphorylation of cofilin in FLT3-ITD+ cells. Combination experiments with LIMKi and FLT3 inhibitors including midostaurin, crenolanib and gilteritinib were synergistic for treatment of MOLM-13 cells while combinations with quizartinib were additive. Combinations of LIMKi and the hypomethylating agent azacitidine or the ROCK inhibitor fasudil were additive. In NOD-SCID mice engrafted with MOLM13-LUC cells, the FLT3 inhibitor midostaurin and LIMKi delayed MOLM13-LUC engraftment as detected by in vivo bioluminescence imaging and the LIMKi and midostaurin combination prolonged significantly survival of leukemic mice. LIMK1/2 inhibition by the small molecule CEL_Amide seems to have promising activity in combination with FLT3 inhibitors in vitro as well as in vivo and may constitute a novel treatment strategy for FLT3-ITD+ AML.

Background: AML is a genetically heterogenous disease with poor prognosis and new treatments are needed. MPS1 is the main kinase of the spindle assembly checkpoint (SAC), critical for segregation of chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. MPS1 inhibition has been investigated in several solid tumors but not yet in AML. Here, we demonstrate the efficacy of the small molecule MPS1 inhibitor (MPS1i) S81694 in AML cell lines, primary cells and mouse models. Materials and Methods: Cell viability, cell stress and apoptosis were assessed by MTS assays, DiOC2(3) or annexin-V modifications and PI incorporation after exposure to S81694. Cell-cycle and polyploidy were determined by cytofluorometry and FISH. Phosphorylation of MPS1 was detected in synchronized cells by immunofluorescence detecting phosphorylated Thr33/Ser37 residues. Protein modifications were studied by WB. Time-lapse microscopy was used to determine mitosis duration using cells stained with the SiR-DNA live cell probe. Bone marrow cells from patients were obtained after informed consent and incubated on a MSC feeder or in methylcellulose for colony formation assays. All animal studies were performed in accordance with the animal ethics committee's guidelines. NSG mice were sub-lethally-irradiated and injected with AML-NS8 or AML-PS patient derived xenografts. Mice were treated intravenously with S81694 or with vehicle. Results: IC50 for viability after S81694 exposure alone was 1000nM: the NPM1cmut cell line OCI-AML3, OCI-AML3-TP53mut and K562. In the most sensitive cell lines, including OCI-AML2, S81694 led to inhibition of MPS1 auto-phosphorylation, induced significant cell stress and apoptosis as detected by mitochondrial membrane potential loss, phosphatidylserine exposure and PI incorporation. In these cell lines, the cell cycle was strongly affected by treatment with S81694 showing aberrant 2n/4n ploidy distribution due to SAC abrogation as well as polyploidy (8n and 16n). S81694 exposure triggered mitotic exit as shown by cyclin B1 downregulation and significantly accelerated mitosis. For treated OCI-AML2 and OCI-AML3 cells we observed induction of gamma-H2AX (ser139), p53 upregulation and downstream caspase-3 and PARP cleavage indicating that S81694 induces p53 dependent apoptosis. Furthermore, MPS1i induced downregulation of anti-apoptotic proteins MCL-1 and BCLXL. Finally, for AML patient derived blast cells cells we observed apoptosis after exposure to S81694 and significant reduction of colony number. In two AML PDX models, intravenous administration of S81694 was shown to improve significantly median survival time, as compared to control, by 28 to 41 days and by 33 to 46 days respectively. Conclusion: The MPS1i S81694 shows significant preclinical activity in vitro and in vivo in AML. Combinations with different drugs active in AML are ongoing. Citation Format: Anna Kaci, Emilie Adicéam, Etienne De Braekeleer, Marine Garrido, Jeannig Berrou, Mélanie Dupont, Hanane Djamai, Virginie Eclache, Baruchel André, Claude Gardin, Hervé Dombret, Mike Burbridge, Thorsten Braun. The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 608.

BACKGROUND The incidence of cancer is increased after solid organ transplantation. Natural killer (NK) cells are key effectors of the tumor immune response. METHODS We conducted a cross sectional multicentre matched case-control study including 42 kidney transplant recipients (KTRs) on diagnosis of cancer and 41 KTRs without cancer. Extensive phenotyping of NK cells populations and functional tests of NK cells were performed. RESULTS Kidney transplant recipients with cancer had a higher incidence of acute rejection (P = 0.02) and cytomegalovirus (CMV) infection (P = 0.03) than controls. They had more lymphopenia than control KTRs (1020/mm3 +/- 32 vs 1218/mm3 +/- 34; P = 0.001) including a CD4+ lymphopenia (P = 0.01). Total CD3-/CD56+ NK cell counts were similar in both groups. However, KTRs with cancer had a lower frequency of the cytokine-enriched CD56bright NK cell subset (P = 0.001). The percentage of NK cells expressing NKp46 was decreased in KTRs with cancer (45% vs 53 %, P = 0.001). Furthermore, the ability of NK cells to degranulate CD107a+ cytolytic vesicles was reduced (11% vs 22%; P = 0.02), and the percentage of NK cells secreting IFN[gamma] was decreased (7.5% vs 28.8%; P = 0.01) in KTRs with cancer. CONCLUSIONS These results reveal an imbalance between NK cell subpopulations and functional NK cell defects in KTRs at the diagnosis of malignancy, including a decreased expression of NKp46 and decreased numbers of NK cells producing INF[gamma]. This study highlights the role of NKp46, a major activating NK cell receptor, which could be considered as a potential marker during immunological follow-up of KTRs.

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.

Bromodomain and Extra-Terminal inhibitors (BETi) such as OTX015 are active in Acute Myeloid Leukaemias (AML). Their activity on Leukemic Stem Cells (LSCs) is less documented. We interrogated the anti-LSC activity of OTX015 in a niche-like long-term culture in 26 primary AML samples and validated our findings in vivo. OTX015 impaired LSCs in AMLs harbouring Core Binding Factor or KMT2A gene fusions, NPM1 or chromatin/spliceosome genes mutations, but not in those with aneuploidy/TP53 mutations. In four patients, we dissected the transcriptomic footprint of Bet inhibition on LSCs versus blasts. Our results can instruct future clinical trials of BETi in AML.

Introduction: The dual-specificity protein kinase, monopolar spindle 1 (Mps1) is one the main kinases of the spindle assembly checkpoint (SAC) critical for accurate segregation of sister chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. Mps1 is known to be overexpressed in several solid tumors including triple negative breast cancer. Thus, Mps1 seems to be a promising target and small molecules targeting Mps1 entered clinical trials in solid tumors. ALL originates from malignant transformation of B-and T-lineage lymphoid precursors with a variety of genetic aberrations including chromosome translocations, mutations, and aneuploidies in genes responsible for cell cycle regulation and lymphoid cell development. While outcome is excellent for pediatric patients and younger adults, relapsed and refractory disease still remain a clinical challenge for elder patients. Here, we demonstrate for the first time preclinical efficacy of the small molecule Mps1 inhibitor (Mps1i) S81694 in T- and B- ALL cells including BCR-ABL1+-driven B-ALL. Materials and Methods: Expression of Mps1 was determined by RT-qPCR and WB in JURKAT, RS4-11 and BCR-ABL1+ cells (BV-173 and TOM-1). A small molecule Mps1i (S81694) was tested alone (0 to 1000nM) or in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1+ ALL cell lines. Cell viability and IC50 was assessed by MTS assays after exposure to Mps1i for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Bliss model. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of Mps1i. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation at 48h. Phosphorylation of Mps1 was detected in synchronized (by nocodazole and MG-132) cells by immunofluorescence using an anti phospho-Mps1 antibody detecting Thr33/Ser37 residues. Time-lapse microscopy was used in cell lines in presence or absence of S81694 to determine mitosis duration. Bone marrow (BM) nucleated patient cells were obtained after informed consent and incubated in methylcellulose with cytokines with or without Mps1i for 2 weeks to determine colony growth. Results: Expression of Mps1 could be detected by RT-qPCR and at the protein level by WB in all cell lines (Figure 1A and B ). IC50 after Mps1i exposure alone was 126nM in JURKAT cells, 51nM in RS4-11 cells, 75nM in BV-173 cells and 83nM in TOM-1. Significant apoptosis as detected by phosphatidylserine exposure and PI incorporation in all cell lines with BCR-ABL1+ cell lines BV-173 and TOM-1 cells being the most sensitive (80% and 60% apoptotic cells respectively)(Figure 1C). Upon Mps1i exposure we observed targeted inhibition of Mps1 phosphorylation at Thr33/Ser37 residues indicating the specific on target effect of S81694 by inhibiting Mps1 autophosphorylation (Figure 1D and E). Cell cycle profile was generally lost after treatment with S81694 in all cell lines indicating aberrant 2n/4n distribution due to SAC abrogation (Figure 1F). Furthermore, we demonstrated that S81694 exposure accelerated significantly mitosis in BV-173 cell line from 36 minutes to 19 minutes indicating effective inhibition of SAC function (Figure 1G). Interestingly, S81694 induced significant apoptosis (70%) in the imatinib resistant BV173 cell line bearing the E255K-BCR-ABL1-mutation. Combination of S81694 with TKI imatinib, dasatinib and nilotinib (but not ponatinib) was strongly synergistic in BCR-ABL1+ cells (Figure 1H). Finally, we observed inhibition of colony formation in a patient with BCR-ABL1+ B-ALL after exposure to 100nM and 250nM S81694 (reduction of 85% and 100% respectively)(Figure 1I). Conclusion: Mps1i S81694 yields significant preclinical activity in T-and B-cell ALL including BCR-ABL1+ models. Interestingly S81694 was efficacious in a TKI resistant cell line. Disclosures Kaci: Institut de Recherches Internationales Servier (IRIS): Employment. Garrido:Institut de Recherches Internationales Servier (IRIS): Employment. Burbridge:Institut de Recherches Internationales Servier (IRIS): Employment. Dombret:AGIOS: Honoraria; CELGENE: Consultancy, Honoraria; Institut de Recherches Internationales Servier (IRIS): Research Funding. Braun:Institut de Recherches Internationales Servier (IRIS): Research Funding.

Introduction: LIM kinases 1/2 are downstream effectors of signalization pathways implicated in cytoskeleton dynamics via phosphorylation of Cofilin family proteins,matrix degradation and in activity control of Aurora kinase A. Recently, Rho kinases (ROCK) were identified to be constitutively activated by FLT3-ITD, BCR-ABL and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon its activation by upstream kinases (ROCK and PAK) LIMK1/2 inactivates Cofilin by phosphorylation, leading to enhanced polymerization of Actin. Here we investigated the potential therapeutic role of LIMK1/2 inhibition in FLT3-ITD mutated AML. Materials and methods: Expression of LIMK1/2 was determined by RQ-PCR and WB. A small molecule inhibitor of LIMK1/2 (LIMKi) was tested alone or in combination with FLT3 inhibitors Midostaurin, Quizartinib and Crenolanib or the hypomethylating agent Azacitidine in FLT3-ITD driven AML cell lines MOLM-13 and MV-4-11. Cell viability and IC50 was assessed by MTT assays. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTT assays and combination index (CI) was calculated with the Chow and Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Apoptosis was evaluated in cell lines and patient cells by outer Annexin V exposure and PI incorporation.Cells from healthy donors were obtained after informed consent and enriched for CD34+ cells by immunomagnetic selection and seeded in methylcellulose, FCS and cytokines with or without LIMKi. For in vivo experiments we used a bone marrow engraftment tumor model with MOLM13-LUC cells using bioluminescence imaging in NOD-SCID mice treated either with LIMKi, Midostaurin or LIMKi+Midostaurin. Results:Expression of LIMK1/2 in MOLM-13 and MV-4-11 cells could be detected by QT-PCR and at the protein level.IC50 after LIMKi exposure was 440 nM in MOLM-13 cells and 420 nM in MV-4-11 cells. Combination experiments with the LIMKi and either the FLT3 inhibitors Midostaurin, Quizartinib, Crenolanib or the hypmethylating agent Azacitidine were synergistic for treatment of MOLM-13 cells. Exposure of MOLM-13 cells to increasing doses of LIMKi induced cell cycle arrest in the G1/S transition and dose dependent apoptosis. No significant toxicity of increasing doses of LIMKi after exposure of CD34+ cells from healthy donors could be detected. In NOD-SCID mice engrafted with MOLM13-LUC cells Midostaurin and LIMKi delayed MOLM13 engraftment as detected by in vivo bioluminescence imaging and LIMKi+Midostaurin prolonged significantly survival of mice as compared to Midostaurin alone. Conclusion: LIMK1/2 inhibition seems to be promising in combination with various FLT3 inhibitors or Azacitidine in vitro as well as in vivo with Midostaurin. Citation Format: Thorsten Braun, Jeannig Berrou, Hanane Djamai, Mélanie Dupont, Anna Kaci, Jan Erik Ehlert, Holger Weber, André Baruchel, Fabrice Paublant, Renaud Prudent, Claude Gardin, Hervé Dombret. Synergy of FLT3 inhibitors and a small molecule inhibitor of LIM kinase1/2 in FLT3-ITD positive acute myeloblastic leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 341.

Immune deficiency is one of the numerous consequences of chronic renal failure. We can hypothesize that an abnormal natural killer (NK) cell response is present in patients with end-stage renal disease (ESRD). To characterize the immune defect in ESRD patients, we performed a NK cell subset analysis in 66 patients with ESRD treated by hemodialysis or peritoneal dialysis. Compared with healthy blood donors, patients undergoing chronic dialysis showed a profound decrease in NKG2D(+) cells within both CD8(+) T cell (58 vs. 67%, p = 0.03) and NK cell (39 vs. 56%, p = 0.002) populations. We further studied NK cell subsets in 55 hemodialysis patients and 17 patients treated by peritoneal dialysis. As compared to healthy donors, all these patients had significantly decreased NKG2D-positive NK cells. Patients using PMMA membranes (BK-F) or Helixone-FX membranes had a smaller decrease in NKG2D-positive NK cells when compared to patients treated with Nephral membranes. The expression of MICA on the cell surface of monocytes, which is a marker of inflammation induced by cellular stress, was also lower in patients using BK-F membranes. In conclusion, these data suggest that a subpopulation of NK cells is decreased in patients with ESRD. This decrease is associated with high circulating levels of the HLA-related molecule MICA. The dialysis membrane can influence the modulation of both the phenotype of NK cells and MICA overexpression.

Introduction: LIM kinases 1 and 2 (LIMK1/2) are downstream effectors at the crossroads of different signaling pathways implicated in the dynamics of the cytoskeleton via phosphorylation of cofilin family proteins, degradation of the matrix by phosphorylating MT1-MMP and control of the activity of Aurora kinase A. Recently, the oncogenic role of Rho kinases (ROCK) was identified to be constitutively activated by BCR-ABL1, FLT3-ITD and KIT in hematologic malignancies via PI3 kinase and Rho GTPase mediated phosphorylation. Upon activation, ROCK phosphorylates LIMK1/2 leading to inactivation of cofilin by its phosphorylation and polymerization of actin and microtubules and possibly to other biological effects mediated by LIMK1/2, not yet fully understood. Here, we demonstrate synergy of a LIMK1/2 inhibitor with BCR-ABL1 tyrosine kinase inhibitors (TKI) in vitro and in vivo in different models for BCR-ABL1 driven ALL. Materials and Methods: Expression of LIMK1/2 was determined by RT-qPCR and WB in cell lines. Phosphorylation of cofilin was detected by WB. A small molecule inhibitor of LIMK1/2 was tested alone and in combination with imatinib, dasatinib, nilotinib and ponatinib in BCR-ABL1 positive ALL cell lines TOM-1 and BV-173. Cell viability and IC50 was assessed by MTS assays after exposure to LIMK1/2 inhibitor for 72h. In combination experiments, compounds were added simultaneously and relative cell numbers were determined at 72h with MTS assays and combination index (CI) values were calculated according to the Chou-Talalay model. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) DNA intercalation. Induction of apoptosis was evaluated by annexin-V exposure and PI incorporation at 72h with increasing doses of LIMK1/2 inhibitor. Peripheral blood (PB) nucleated cells from apharesis products of healthy donors obtained after informed consent according to Helsinki declaration were incubated with or without LIMK1/2 inhibitor for 72h, and then enriched for CD34+ cells by immuno-magnetic selection and seeded in triplicate in methylcellulose FCS and cytokines. In vivo experiments were performed in C57Bl/6 mice injected with BCR-ABL-induced B-ALL cells. These were obtained by transduction of CDKN2A-deficient B-cell progenitors with a retrovirus coding for BCR-ABL1 (P185) and GFP, followed by transplantation in sub-lethally-irradiated recipient C57Bl/6 mice. Mice were treated either with LIMK1/2 inhibitor, nilotinib or the combination of both and compared to untreated control mice. Results: Expression of the two isoforms LIMK1 and LIMK2 in TOM-1 and BV-173 cells could be detected by RT-qPCR and at the protein level by WB. IC50 after LIMK1/2 inhibitor exposure alone was 580nM in TOM-1 cells and 1000nM in BV-173 cells. All combination experiments with the LIMK1/2 inhibitor and imatinib, dasatinib, nilotinib and ponatinib yielded synergistic CI for treatment of both TOM-1 and BV-173 cell lines. Cell cycle arrest in the G1/S transition was detected and LIMK1/2 inhibition induced dose dependent apoptosis in TOM-1 and BV-173 cells up to 40% at doses 1% GFP+ cells were detected in PB. Furthermore, nilotinib+LIMK1/2 inhibitor prolonged significantly the survival of mice compared to either nilotinib (p=0.0006) or LIMK1/2 inhibitor alone and untreated mice (p Conclusion: Combination of LIMK1/2 inhibitor with BCR-ABL targeting TKI is synergistic and has significant anti-leukemic activity in BCR-ABL1+ ALL in vitro and in vivo models. Disclosures Braun: CELLIPSE: Research Funding. Prudent:CELLIPSE: Employment. Paublant:CELLIPSE: Employment. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Shire: Research Funding; Servier: Consultancy; Amgen: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Roche: Consultancy. Dombret:CELLIPSE: Research Funding.

Background: Differentiation based therapy by all trans retinoic acid (ATRA) and arsenic trioxide (ATO) results in cure of >90% of patients with acute promyelocytic leukemia (APL). ATRA+ATO is highly biologically active in NPM1c AML, accounting for 30-40% of AML patients. ATO/ATRA induces proteasomal degradation of NPM1c, differentiation, growth arrest and TP53 dependent apoptosis in NPM1c cells. Furthermore, ATRA/ATO exposure restores nuclear localization of NPM1wt and significantly reduces blasts in NPMc AML patients. It was shown that the BET inhibitors OTX015/MK-8628 and JQ1 yield antileukemic activity and here we demonstrate their effects in NPM1c leukemia cells compared to ATRA/ATO. Methods : NPMc OCI-AML3 cell line or patient bone marrow (BM) blast cells obtained after informed consent were exposed to ATRA/ATO or OTX015/MK-8628 and JQ1. Apoptosis was assessed by annexin V/PI and caspase 3/PARP cleavage by WB. TP53 expression was detected by WB. Knock down of TP53 was performed with siRNA. Differentiation of OCI-AML3 cells was studied by CD11b surface expression and morphologic studies after MGG stain. Gene expression profiling was performed with GeneChip Array (Affymetrix®). NPMc expression was assessed by WB (+/- bortezomib) and cellular localization of NPMc/NPMwt was studied by immunofluorescence. Results : Exposure of OCI-AML3 cells to OTX015/MK-8628 and JQ1 was more potent to induce apoptosis as compared to ATRA/ATO. All treatments lead to caspase 3 and PARP cleavage. In OCI-AML3 cells, ATO-ATRA induced strong upregulation of genes of the TP53 dependent pathway (BAX/GADD45) while the anti apoptotic gene BCL2 was downregulated. In contrast, treatment with BET inhibitors lead to strong down regulation of the TP53 dependent pathway. In line, ATRA/ATO induced TP53 protein expression and TP53 knock down by siRNA decreased significantly ATRA/ATO induced apoptosis suggesting that apoptosis induced by BET inhibitors is TP53 independent. As compared to ATRA/ATO, OTX015/MK-8628 and JQ1 were more potent to induce differentiation as detected by CD11b surface expression and by morphologic analysis of OCI-AML3 cells. Interestingly, gene expression profiling of human leukocyte differentiation pathways in OCI-AML3 cells revealed different expression profiles for exposure to BET inhibitors compared to ATRA/ATO. Treatment of OCI-AML3 cells either by OTX015/MK8628, JQ1 or ATRA/ATO lead to proteosomal degradation of the NPMc protein. Exposure of OCI-AML3 cells and primary BM blasts of patients either by OTX015/MK8628, JQ1 or ATRA/ATO led to nuclear relocalization of NPMwt protein to the nucleus. Conclusion : BET inhibitors induce TP53 independent apoptosis, differentiation, proteasomal degradation and NPMwt relocalization in NPMc cells. Thus, clinical testing of bromodomain inhibitors in NPMc AML is indicated. Citation Format: Thorsten Braun, Marie-Magdelaine Coudé, Jeannig Berrou, Hanene Djamai, Mélanie Dupont, Anna Kaci, Marc Delord, Raphael Itzykson, Emmanuel Raffoux, Caroline Berthier, Hugues de Thé, André Baruchel, Claude Gardin, Hervé Dombret. Bromodomain and extra-terminal BET inhibitors induce TP53 independent apoptosis, maturation and oncoprotein degradation in NPM1 mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 803.

Type 1 plasminogen activator inhibitor (PAI-1) is the primary inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Whereas PAI-1 is not expressed in normal kidneys, it is strongly induced in glomerular diseases and thus could promote the local accumulation of fibrin. To study the role of PAI-1 in the development of inflammatory glomerular injury, passive antiglomerular basement membrane (GBM) glomerulonephritis (GN) was induced in PAI-1 knockout mice and in wild-type mice of the same genetic background. Unexpectedly, PAI-1 deficiency was associated with an early and severe exacerbation of glomerular injury: Infiltration by CD4 T cells, proportion of fibrinous crescents, and renal function impairment were significantly more pronounced in PAI-1 -/- mice. Interestingly, activation of transforming growth factor (TGF)- beta, which is known to be dependent on the PA/plasmin system in vitro, was dramatically enhanced in the kidneys in the absence of PAI-1. Moreover, administration of neutralizing antibodies against TGF-beta significantly attenuated the disease in PAI-1 -/- mice. This suggests that the poor outcome of GN in PAI-1 -/- mice is consecutive to an uncontrolled activation of TGF-beta and confers PAI-1 with a new, immunomodulatory role.

The urokinase receptor (uPAR) is a multifunctional molecule involved in pericellular, fibrinolytic, and proteolytic activities, as well as in cell adhesion and chemotaxis and may play a role in the pathogenesis of tissue remodeling occurring during glomerulonephritis. We analyzed sequentially the expression of uPAR by immunohistochemistry and in situ hybridization in an accelerated model of nephrotoxic nephritis in rats. A strong induction of uPAR mRNA expression was observed in glomeruli as soon as 1 h after nephrotoxic serum injection. The intensity of glomerular uPAR mRNA and antigen expression increased and peaked at 24 h. At that time, numerous glomerular fibrin deposits, monocyte/marcrophage infiltration, and heavy proteinuria were observed. Fibrin deposition was detected at 6 h, peaked at 24 h, and progressively declined over the next 3 weeks, while uPAR antigen expression remained elevated until the end of the study (3 weeks). By double labeling, we showed that the expression of uPAR was mediated by both intrinsic glomerular cells and infiltrating macrophages. Severe podocytic lesions developed within 3 days after antiserum injection, and glomerulosclerosis rapidly progressed within 2–3 weeks. These results show that glomerular uPAR expression is induced in nephrotoxic nephritis and suggest that uPAR may promote local proteolysis and also tissue remodeling, leading to the late development of glomerulosclerosis.

BACKGROUND Plasminogen activator inhibitor type 1 (PAI-1) exerts antifibrinolytic and profibrotic activities. Inside the glomerulus, PAI-1 is mainly synthesized by mesangial cells. We hypothesized that thrombin, via its receptor protease activated receptor type 1 (PAR-1), present on the membrane of glomerular cells, is an important mediator of PAI-1 synthesis. METHODS Using the technique of Peten et al., we microdissected the glomeruli of 23 kidney transplanted patients admitted in our department from 1993 to 1997, and we followed-up these patients for up to 5 years, with sometimes iterative renal biopsies. With this technique, we also microdissected the glomeruli of three patients who have had a nephrectomy for cancer (control patients). We investigated mRNA expression of the PAI-1, the thrombin receptor PAR-1, the alpha2 chain of type IV (alpha2 IV) collagen, and of a housekeeping gene (cyclophilin) by reverse transcription-polymerase chain reaction. The results were correlated with the renal function and the histological findings classified into acute rejection (9 biopsies), chronic rejection (22 biopsies), or normal (8 biopsies). RESULTS A significant up-regulation of PAI-1 and alpha2 IV collagen mRNA was observed in acute rejection (P

Hyperfiltration, associated with kidney hypertrophy and hyperplasia, are early markers of diabetic nephropathy [1, 2]. Procedures for the identification of renal molecules potentially involved in this early process are of particular interest. In order to detect hyperexpression of major renal proteins in early diabetic nephropathy, we used the well-known model of streptozotocin (STZ)induced diabetes in rats [3] and a robust technique of subtracted hybridization between normal and diabetic kidneys [4 ‐ 6]. Two subtracted probes were elaborated after hydridization of diabetic rat kidney cDNA with control rat kidney mRNA. These probes were used to screen a diabetic rat kidney cDNA library. Two clones were overexpressed in diabetic rat kidneys and corresponded to the multi-drug resistance 1 (mdr 1) cDNA and to the b-amyloid protein precursor (bPP), respectively. This study describes the results of subtractive hybridization cloning in rats, as well as some data obtained in human kidney sections originating from diabetic patients.

Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen. Some proteases possess a membrane receptor that focalizes their proteolytic activity on the cell surface and may mediate a proliferative effect, such as urokinase on glomerular epithelial cells. Since some hypertensive states are associated with high concentrations of renin and proliferation of arteriolar smooth muscle cells, we asked whether renin, an aspartyl-protease, would bind to mesangial cells that are smooth-muscle derived cells, which would induce their proliferation. The binding of 125 I labeled recombinant human renin ( 125 I-R) was studied on human primary mesangial cells and mesangial cells immortalized by transfection with SV40-T antigen. At 37°C, the binding of 125 I-R was time dependent and reached a plateau after two hours. 125 I-R was found to bind in a saturable and specific manner with a K d = 0.4nM and 1nM and 8,000 and 2,000 binding sites/cell, for primary and immortalized cells, respectively. When binding experiments were performed in the presence RO 42-5892, a synthetic inhibitor of renin, RO 42-5892 could inhibit the specific binding of labeled renin only at concentrations 1,000 times superior to the IC 50, indicating that the renin-mesangial receptor interaction did not depend on the active site of renin. Analysis by SDS-PAGE and autoradiography of cross-linking experiments of 125 I-R bound to a membrane preparation showed a band of approximately 110 to 120 kDa, suggesting a Mr of 70 to 80kDa for the renin receptor. Incubation of mesangial cells with 100nM renin for 24 hours provoked a 100% increase of 3 H thymidine incorporation that was not accompanied by an increase of the cell number, even after a seven day period of incubation. However, the incubation of mesangial cells with renin for 24 hours induced a significant increase (170% of control, P = 0.04) of plasminogen activator inhibitor-1 (PAI1) antigen in the conditioned medium. In conclusion, we have shown that human mesangial cells in culture express a specific receptor for renin, and that the binding of renin increases 3 H thymidine incorporation independently of renin enzymatic activity. The absence of cell proliferation, the increase of 3 H thymidine incorporation and the increase of PAI1 antigen suggest that the binding of renin can induce mesangial cell activation, which is reflected by a change in the fibrinolytic capacity of the cells. The role of this receptor remains to be determined in nephropathies and hypertensive states associated with high plasma/tissue renin concentrations, hypertrophy of mesangial or smooth muscle cells and extracellular matrix remodeling.

Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P

Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Long-term T-cell reconstitution after hematopoietic stem cell transplantation (HSCT) is dependent on patient thymic function and affected by graft-versus-host disease (GVHD). To assess the impact of acute GVHD (aGVHD) on thymic function, we followed a cohort of 93 patients who received HSCT from a human histocompatibility leukocyte antigen-identical sibling, mainly for hematologic malignancies. Thymic output was measured by signal-joint T-cell receptor excision circles (sjTREC) real-time polymerase chain reaction. Absolute sjTREC number was lower at 6 months in patients with aGVHD (P = .014), associated with lower absolute counts of naive CD4 T cells at 6 and 12 months (P = .04 and .02), and persistent abnormalities in T-cell repertoire diversity. Age and aGVHD affected thymic function independently in multivariate analysis. In patients less than 25 years of age, thymic function recovered almost totally at 1 year. As a marker of thymocyte proliferation, we quantified the βTREC generated during the T-cell receptor β-chain recombination, in a group of 20 age-matched patients. Mean βTREC level was reduced at 6 months in patients with aGVHD, indicating an impact on early thymic differentiation rather than on intrathymic proliferation. These data show that aGVHD or its treatment has a transient impact on thymic function in younger patients in the first months after HSCT.

To characterize the immune defect of patients with end-stage renal disease (ESRD), we performed NK cell subset analysis in 66 patients with ESRD treated by hemodialysis (n = 59) or peritoneal dialysis (n = 7). Compared with healthy blood donors, patients undergoing chronic dialysis showed a profound decrease in NKG2D+ cells within both the CD8+ T cell (58% vs 67%, p = 0.03) and NK cell (39% vs 56%, p = 0.002) populations. CD56dim cells, which comprise the majority of NK cells in the periphery, were more affected in this regard than were CD56bright cells. Uremic serum could decrease NKG2D expression on NK cells from healthy donors. Among factors that could contribute to the decrease in NKG2D expression in ESRD patients, reactive oxygen species (ROS) play a major role. We found that catalase could reverse the effects of uremic serum on NKG2D expression (p < 0.001) and that ROS down-regulated NKG2D at the mRNA level and at the NK cell surface. Additionally, ESRD patients had both increased membrane-bound MHC class I-related chain A (MICA) on monocytes (p = 0.04) and increased soluble MICA (203 pg/ml vs 110 pg/ml; p < 0.001). Both ROS and uremic serum could significantly increase in vitro the expression of the NKG2D ligand MICA on the renal epithelial cell line HK-2. Taken together, these studies suggest for the first time that both low NKG2D expression and up-regulation of its ligand MICA are related to ROS production and may be involved in the immune deficiency of ESRD patients.

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: 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.

The activation of the protease-activated receptor 1 (PAR-1) by thrombin has been shown to induce an activation of the MAP kinase cascade and to stimulate cell proliferation. To examine the mechanisms of signal transduction by PAR-1, we constructed several PAR-1 mutants which were stably expressed in CHO cells. When compared to wild-type PAR-1, mutation of Ser306-->Ala (S306A) in the third intracellular loop of PAR-1 inhibited MAP kinase activation and cell proliferation stimulated by thrombin. The thrombin activation of MAP kinase was inhibited by pertussis toxin, suggesting a role for a Gi-like protein. As shown by calcium signaling and inosotol trisphosphate generation, the Ser306-mutated PAR-1 induced a strong activation of phospholipase C after thrombin addition. Deletion of the cytoplasmic tail of PAR-1 also inhibited thrombin-induced DNA synthesis but the MAP kinase pathway was activated as with wild-type PAR-1. In contrast, the deletion of the C-tail of PAR-1 prevented almost completely the activation of the phospholipase C pathway. Taken together these results suggest that the C-tail of PAR-1 is a critical site for PAR-1 coupling to phospholipase C activation, while the third intracellular loop of PAR-1 is implicated in PAR-1 coupling to Gi and MAP kinase activation. In addition, these results also show that MAP kinase activation is necessary but not sufficient for thrombin to induce cell proliferation.

To analyse the mechanisms of PAR-1 internalisation, we constructed several PAR-1 mutants and stably expressed them in CHO cells. Our study shows that the Ser(306)--Ala mutation (S306A), which eliminates a potential site of phosphorylation by PKC in the third intracellular loop of PAR-1, did not change the rate of phosphorylation but reduced the rate of thrombin-induced internalisation of the PAR-1 mutant (58 versus 78% of membrane PAR-1 in 15 min, p0.005). Deletion of the last 43 amino acid residues of the PAR-1 cytoplasmic tail completely suppressed the thrombin phosphorylation of the mutated receptor and significantly reduced its internalisation upon activation. This deletion also inhibited the PMA-induced and the agonist-independent internalisation of the receptor. The Tyr(371)--Ala mutation (Y371A), in a NPXXY motif of the seventh transmembrane domain of the receptor had no effect on the receptor behaviour. Our results indicate that both the C-tail and the third intracellular loop are involved in PAR-1 internalisation induced by thrombin while only the C-tail plays a role in the PMA-induced and in the agonist-independent PAR-1 internalisation.

thrombocytopenia and ischemic lesions leading to either renal impairment in the case of HUS, or neuroBackground. In children, haemolytic uraemic syndrome (HUS ) is associated with high plasma plasmin- logical dysfunction in the case of thrombotic thrombocytopenic purpura ( TTP) [1]. HUS is a consequence ogen activator inhibitor type 1 (PAI-1), which may contribute to the persistence of renal glomerular and of endothelial cell injury which is mediated by Shigaarteriolar thrombi. HUS has been described in HIV- like toxins produced by enterobacteria [2], exposure infected patients, but the pathophysiology of HUS in to various drugs or possibly anti-endothelial cell these patients is poorly understood. The aim of the autoantibodies [3]. Besides having anticoagulant propstudy was to investigate plasma fibrinolytic activity in erties, endothelial cells prevent platelet aggregation 18 patients with HIV-associated HUS. and stimulate fibrinolysis. They are the main source of Methods. We measured tissue type plasminogen activ- tissue-type plasminogen activator (t-PA) in the blood ator (t-PA) and PAI-1 activities in the plasma of 18 and can also secrete PAI-1, the physiological inhibitor HIV-infected patients with biopsy-proven HUS of t-PA [4]. Recently, two studies performed in children (HIV+/HUS+) and 48 HIV-infected patients without with HUS [5,6 ] reported that plasma PAI-1 was HUS (HIV+/HUS’). increased and may be of prognostic value in the longResults. Patients with HUS had a significantly higher term renal outcome. serum creatinine, a lower platelet count and an More than 50 cases of HUS have been described in increased incidence of cytomegalovirus (CMV ) infec- adult patients with HIV infection [7,8] but little is tion (72% of patients HIV+/HUS+, vs 25% of known about the link between HUS and HIV infection. patients HIV+/HUS’). Unexpectedly, plasma PAI-1 Recently, we described 29 cases of HUS in HIVactivity was similar in both groups. However, t-PA infected adult patients [9], most of them with biopsyactivity was significantly higher in HUS cases (11.5 vs proven HUS. Clinical data, pathological analysis and 4.5 U/ml, P=0.001). Patients with CMV infection, a retrospective case-control study lead to the concluwith or without HUS, had significantly increased t-PA sion that HUS was a late complication of HIV infection level (P=0.01). Multivariate analysis identified high and was associated with systemic cytomegalovirus t-PA (RR=9.21) and CMV infection (RR=3.36) as (CMV ) infection. We report here that plasma t-PA risk factors for HUS. activity is increased in HIV-infected patients with HUS, Conclusion. This study provides evidence that HIV- especially in the group of patients with active CMV infected patients with HUS have high plasma t-PA infection. Plasma PAI-1 activity is only slightly activity. PAI-1 plasma activity is not significantly modified, suggesting that high PAI-1 plasma level may increased, as opposed to non-HIV-associated HUS. not explain the persistence of fibrin deposits in Thus, in the setting of HIV infection, HUS cannot be HIV-related HUS. attributed to decreased fibrinolytic activity.

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.

Endothelin-1 acts as a potent autocrine and paracrine mediator in the kidney. Glomeruli and renal arterioles also express functional thrombin receptors which mediate the cellular effects of thrombin. Using the binding on glomerular epithelial cells of 125I-labeled ATAP2, a monoclonal antibody raised against the functional thrombin receptor, we demonstrate here that endothelin-1 induces thrombin receptor internalization in a dose- and a time-dependent manner. The maximal effect is observed at 10(-7) M endothelin-1 corresponding to internalization of approximately 25% of thrombin receptors. It is maximal at 20 min and persists for at least 24 h. This effect is mediated by the endothelin receptor subtype A (ETA), and involves a pertussis toxin-sensitive G-protein and protein kinase C activation. Endothelin-1 does not induce thrombin receptor degradation nor change in thrombin receptor mRNA level after 2 h and 24 h of incubation. These results indicate that partial heterologous internalization of the functional thrombin receptor is induced by endothelin-1.

Sezary syndrome (SS) is a rare lymphoma characterized by the clonal expansion in the skin and in blood of CD4(+)CD158k(+) T cells. Natural killer (NK) activation against tumors in leukemia models is partly based on the recognition of the target through the NKG2D/NKG2D ligands interactions. We analyzed ex vivo SS malignant lymphocytes for the expression of the NKG2D ligands such as the major histocompatibility complex class I-related molecules (MIC) A and B and the UL16 binding proteins (ULBP). The expressions of NKG2D, the natural cytotoxicity receptors (NKp30, NKp44, and NKp46) and the activating receptor DNAM-1 were simultaneously investigated on circulating patients NK and CD8(+) nonmalignant lymphocytes. Interestingly, although at least one of the NKG2D ligands was expressed on the circulating malignant lymphocytes of 9 out of 10 patients, NKG2D was expressed on effector lymphocytes. We found that soluble MICA in patient's sera was increased, which may constitute a mechanism to escape the immune response. In vitro, SS tumor lymphocytes induced the degranulation of perforin by the NKL cell line. More importantly, NKG2D expressed on SS patients NK cells is functional and capable to induce degranulation. Altogether, these data could suggest that stimulating NK function in SS patients may be a promising strategy to reduce tumor invasion.