ALBERT

Description: Despite a high rate of complete remission after treatment with conventional genotoxic agents, the overall survival of patients with acute myeloid leukemia (AML) is poor due to frequent relapses caused by the chemoresistance of rare leukemic stem cells (LSCs, also called Scid-Leukemia Initiating Cells). This unfavorable situation leads to a strong need to characterize those cells in order to target them with new specific therapies. Using a robust immunodeficient mouse model (NOD/LtSz-scid IL2Rγchainnull or NSG), we have previously shown that these LSCs were rare and not restricted to the CD34+CD38- immature compartment. This phenotypical heterogeneity of LSCs suggests that pharmacological targeting of LSC will not work if solely based on their cell surface markers. A better understanding of the mechanisms underlying the in vivo chemoresistance is required for the development of innovative targeted therapies. Aracytine (Ara-C, a pyrimidine analog), the most clinically used chemotherapeutic agents for AML patients, inhibits DNA synthesis and, therefore, targets and kills cycling AML cells in S phase of the cell cycle. Based on this mechanism of action, we hypothesized that Ara-C treatment will spare and enrich quiescent LSCs in vivo. We analyzed the response to Ara-C and residual disease in NSG mice engrafted with primary AML cells from 13 patients in two clinical centers (University of Pennsylvania, Philadelphia, USA and Purpan Hospital, Toulouse, France). A sub-lethal treatment of 60 mg/kg Ara-C given daily for five days induced a 5- to 50- fold reduction of peripheral blood blasts and total tumor burden in spleen and bone marrow in all patients tested. For 5 patients, we observed relapse within 4 to 6 weeks post-chemotherapy. Surprisingly, residual viable cells after Ara-C treatment showed no significant enrichment in quiescent cells and CD34+CD38- cells for the majority of primary samples tested (12 and 10 out of 13 total tested, respectively). Of note, the largest fraction (70-90%) of leukemic cells is in G0/G1 phase (including 0.5-20% in G0) in untreated engrafted mice. Moreover, we observed no significant changes in cell cycle profile of residual leukemic cells during the time course of the disease progression for 3 out of 4 patients. Finally, we assessed the frequency of LSCs in Ara-C-treated and control mice using transplantation and limiting dilution analysis in secondary recipients. Interestingly, we observed that Ara-C treatment did not increase the frequency of SL-ICs in residual cells, suggesting that blasts and LSC were equally sensitive to Ara-C in vivo. Our results show that sub-lethal regimen of Ara-C does not lead to enrichment of LSCs and induces cell death of both leukemic bulk and stem/progenitor cells independently of their cell cycle status probably through another in vivo mechanism such as apoptosis, autophagy or necroptosis. This study also suggests that further characterization of chemoresistant leukemic cells beyond phenotype and cell cycle status must rely on more functional properties in order to better elucidate the molecular basis of resistance in AML. Disclosures: Perry: MERCK: Employment. Carroll:Leukemia and Lymphoma Society: Research Funding. Sarry:AFFICHEM SA: Membership on an entity’s Board of Directors or advisory committees.

Description: White adipose tissue (WAT) is the focus of new interest because of the presence of an abundant and complex immune cell population that is involved in key pathologies such as metabolic syndrome. Based on in vivo reconstitution assays, it is thought that these immune cells are derived from the bone marrow (BM). However, previous studies have shown that WAT exhibits specific hematopoietic activity exerted by an unknown subpopulation of cells. In the present study, we prospectively isolated a peculiar hematopoietic stem/progenitor cell population from murine WAT. The cells are phenotypically similar to BM hematopoietic stem cells and are able to differentiate into both myeloid and lymphoid lineages in vitro. In competitive repopulation assays in vivo, they reconstituted the innate immune compartment in WAT preferentially and more efficiently than BM cells, but did not reconstitute hematopoietic organs. They were also able to give rise to multilineage engraftment in both secondary recipients and in utero transplantation. Therefore, we propose that WAT hematopoietic cells constitute a population of immature cells that are able to renew innate immune cell populations. Considering the amount of WAT in adults, our results suggest that WAT hematopoietic activity controls WAT inflammatory processes and also supports innate immune responses in other organs.

Description: Resistant cells to conventional chemotherapy - which targets DNA synthesis - are thought to be rare and enriched in quiescent leukemic stem cells (LSCs) in acute myeloid leukemia (AML). In order to develop an improved understanding of chemotherapy resistance in AML, we analyzed the response to cytarabine (Ara-C) in our xenograft model with 20 primary AML patient specimens from two clinical sites. After demonstrating AML engraftment, mice are treated with Ara-C given IP daily for 5 days as a single agent at 60 mg/kg daily, which correlates with human dosing. In all mice treated with this regimen, there was a 4- to 46-fold cytoreductive effect at 1 week post-treatment. While in vivo Ara-C treatment induces changes in CD34pos CD38pos/neg phenotypes of 50% of AML-engrafted mice, this treatment does not induce any significant changes in cell cycle distribution and does not enrich in G0 quiescent cells in AML-engrafted mice. Surprisingly we observed that Ara-C induces a decrease or no change in the frequency of SL-ICs (control, 1:2,500-21,900 versus Ara-C, 1:4,500-34,200), suggesting that Ara-C would not spare LSCs in vivo and equally kills both cycling and quiescent cells. Furthermore, transcriptomic analysis of Ara-C residual leukemic cells (RLCs) from three different patient-derived xenografts confirmed that RLCs are not enriched in stem cell marker genes at 1 week post-treatment. However, our gene signature of the in vivo AML resistance is enriched in genes involved in inflammatory, immune and stress response. When tested in three independent AML cohorts (Verhaak et al. 2009; TGCA, 2011; Metzeler et al. 2011), this signature is associated with an unfavorable prognosis in patients treated with intensive chemotherapy. Finally we have shown that RLCs are PIMOpos cells with both high ROS content and active mitochondrial mass and membrane potential in AraC-treated xenografted mice compared to control mice. Accordingly, AML cells exhibiting high OXPHOS energetic phenotype are resistant to Ara-C chemotherapy in vivo while low OXPHOS AML cells are sensitive to AraC in NSG mice. Altogether these results suggest a novel model of cytarabine chemotherapy resistance in AML based on the oxidative and mitochondrial energy metabolism in vivo. Disclosures No relevant conflicts of interest to declare.

Description: Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, amongst which IDH mutations occur in 15% of AML patients. Here, we show both in vitro as well as in a xenografted mouse model, that clinically achievable doses of ATRA are sufficient to achieve a terminal granulocytic differentiation in primary AML samples and in AML cell lines harboring IDH1-R132H mutation. There is no effect at this concentration on the WT controls. This is associated with reduction of both proliferation and colony formation, and further leads to apoptosis, thereby improving overall survival of mutant xenografted mice. We further showed, through transcriptomic and western blot analysis, that specific ATRA sensitivity is due to overexpression and activation of C/EBPα in the presence of IDH1-R132H mutation. This primes blasts into myeloid differentiation. Moreover, IDH1 R132H mutation also reduces LYN activation, and thus, also sensitizes to clinically achievable doses of dasatinib, a LYN inhibitor. As ATRA induces a brief LYN activation, which transiently reduces ATRA activity, its combination with dasatinib synergistically increases differentiation. In vivo, the combination of ATRA and dasatinib reduces tumor growth of mutant xenografted mice. The combination ATRA and dasatinib might also be considered for other IDH mutations that produce 2-hydroxyglutarate, since treatment with the mutant-specific oncometabolite (eg. 2-hydroxyglutarate) sensitizes AML cells to ATRA and dasatinib-induced differentiation. Finally, ATRA also reduces BCL2 expression specifically in the presence of IDH1 R132H mutation. Since it has been shown that IDH mutations increase BCL2 dependence in leukemic cells, our results identified a subgroup of patients that is likely to respond to pharmacologic concentrations of ATRA. To conclude, our data provide the preclinical rationale for investigating the use of the combination ATRA and dasatinib in a subgroup of patients who carry IDH1 R132H mutation, in clinical trials. The addition of a BCL2 inhibitor such as ABT-199 would also be considered. Disclosures Off Label Use: ATRA and dasatinib for treatment of non APL AML. Recher:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Chugai: Research Funding.

Description: Acute Myeloid Leukemia (AML) is the most common type of leukemia in adults. Despite intensive research, current treatments remain unsatisfactory with only 40% of younger (60 years) AML patients achieving long-term complete remission. Consequently, drugs with novel mechanism of action are urgently needed to improve the outcome of these patients. We have recently identified Dendrogenin A (DDA) as a cholesterol metabolite present in normal cells but undetectable in various cancer cell lines including AML (de Medina et al, Nat Commun, 2013). DDA, the first steroidal alkaloid identified in mammals, exhibited strong anticancer effects against different tumor models in vitro and in vivo. In this study, we investigated the antileukemic potency of DDA in AML. We demonstrated that DDA exerts potent cytotoxic effect in a large panel of AML cell lines and cytogenetically and molecularly diverse primary AML patient samples (n=50) with a median IC50 of 3.3 µM (range 1.2-10 µM). We determined that DDA triggers both apoptosis and cytotoxic autophagy on AML cells. Macroautophagy was characterized by the accumulation of autophagic vacuoles and the stimulation of autophagic flux. As opposed to conventional chemotherapies, the antileukemic effect of DDA was similarly efficient in both immature stem/progenitor CD34+CD38-CD123+ subpopulation and leukemic bulk. Interestingly, the antileukemic activity of DDA on AML patient samples was not correlated to usual prognostic factors such as adverse cytogenetic risk karyotype, clonogenic ability, white blood cells count and FLT3-ITD or NPM status. Pharmacokinetic studies revealed that both per os (PO) and intraperitoneal (IP) administration led to a good absorption with calculated bioavailability of 74% (PO) and 48% (IP), showing that these modes of administration are relevant to in vivo preclinical studies. We then examined the in vivo anti-leukemic efficacy of DDA in NOD/SCID mice injected subcutaneously with HL60 and KG1 cells. We demonstrated that daily administration of DDA (20 mg/kg IP or 40 mg/kg PO) significantly reduced KG1 and HL60 tumor growth. Immunohistochemical analysis revealed that AML xenografts from mice exposed to DDA display a 3.5 fold increase of LC3 punctated cells and a decreased P62 level highlighting that DDA induces autophagy in vivo. Furthermore, DDA significantly kills AML cells in bone marrow and brain (55±5.6% reduction of viable CD45+ cells), and strongly reduces (57±7.8%) the total cell tumor burden in bone marrow and spleen in established disease models (eg. orthotopically engraftment of HL60 cells and three primary AML patient cells via tail vein injection in NOD/SCID/IL2Rγc-deficient mice). In addition, we showed that DDA is well tolerated in mice at effective dose and spares normal hematopoietic stem/progenitor cells from healthy donor. Mechanistic studies revealed that DDA is a natural modulator of the Liver X Receptor (LXR), a nuclear receptor involved in cholesterol homeostasis, immunity and proliferation. We found that the silencing of LXRβ gene prevents the capacity of DDA to trigger both cell death and autophagy on AML cells in vitro. In addition, DDA failed to block tumor development and to trigger autophagy on LXRβ-invalidated KG1 cells xenografted on NOD/SCID mice. Moreover, DDA strongly stimulates the expression of the myeloid leukemogenesis tumor suppressors Nur77 and Nor1 through an LXRβ-dependent mechanism. Interestingly, DDA triggers the relocation of Nur77 to the mitochondria, a process associated with both apoptosis and autophagic cell death. This study provides a strong rationale to bring DDA in clinical trials for patients with AML. Disclosures de Medina: Affichem: Employment. Bize:Affichem: Employment. Paillasse:Affichem: Employment. Noguer:Affichem: Employment. Sarry:Affichem: Equity Ownership. Silvente-Poirot:Affichem: Equity Ownership. Poirot:Affichem: Equity Ownership.