ALBERT

Description: Background:Acute promyelocytic leukemia (APL) is a favorable-risk subgroup of AML characterized by the t(15;17) translocation. The leading cause of early death in APL is uncontrolled bleeding mostly attributed to aberrant expression of tissue factor (F3) and annexin A2 (ANXA2) on leukemic promyelocytes leading to disseminated intravascular coagulation and hyperfibrinolysis, respectively. To prevent or treat such complications, early suspicion of APL and rapid initiation of therapy and supportive measures are critical. Podoplaninor PDPN is a surface glycoprotein expressed in most cell types (http://www.gtexportal.org), but not in blood cells. CLEC-2, the PDPN receptor, is expressed on normal platelets and was found to be necessary for the separation of blood and lymphatic vessels during embryogenesis. PDPN expression (whether endogenous or ectopic) in cell lines induces platelet aggregation, which can be inhibited by chemical tool compounds or by monoclonal antibodies (Chang et al, Oncotarget, 2015, Kato et al, Biochem. Biophys. Res. Commun., 2006). Aims and Methods:We analyzed the transcriptome of 30 APL comprised in the Leucegene 430 AML cohort, aiming to identify clinically useful markers and to better understand the hemostasis-related transcriptomic landscape of this subgroup. Patient cohorts and sorted normal hematopoietic cell populations (n=63) were previously reported (Lavallée et al, Nature Genetics, 2015 and Lavallée et al, Blood, 2016). Comparative analysis of gene expression and mutations were performed as previously described. Results:Our analytical pipeline identified several mutated genes in this cohort, most of which are non-specific and previously identified. CEBPE mutations were the only exception and were specific to APL specimens in this cohort (2/30 vs 0/400, p= 0.005). Most interestingly, we identified PDPN as the single most differentially overexpressed gene in APL (median 2.6 vs 0 RPKM, q = 7 x 10-29, Fig A-B). We also found that PDPN is not expressed in whole blood, bone marrow and in any sorted cell subpopulations from these normal tissues, including promyelocytes (median PDPN expression = 0 RPKM, range 0-0.018). This indicates that platelets are never exposed to PDPN in the adult vasculature and reveals that this gene is ectopically expressed in APL promyelocytes. Accordingly, our hypothesis is that aberrant PDPN expression on leukemic promyelocytes contributes to abnormal platelet aggregation in APL patients. We found that high PDPN expression is associated with lower platelet counts at presentation (18 vs 34 x 1012/L, median PDPN expression ≥ 10 vs 〈 10 RPKM, p = 0.016, Fig C). Furthermore, a strong inverse correlation was observed between the number of estimated circulating PDPN+ promyelocytes and platelet counts (leukocyte count x [% PDPN+ cells] 〉 1 x 109/L, Fig C, p=0.007). By incorporating anti-PDPN antibody (clone NC-08, Biolegend) in the EuroFlow protocol, we observed that PDPN expression test was 90% sensitive and 100% specific for APL (n= 48 and 50 APL and non-APL primary AML, respectively). Of note, 5 APL cases considered positive expressed low levels of PDPN. Interestingly, by comparing expression of all coagulation and fibrinolysis genes in APL (n=30) to that of non-APL specimens (n=400), we found that PDPN was the most discriminatory transcript (Fig. A). This result stands in sharp contrast with that found with F3 and ANXA2 which largely overlap in these APL versus non-APL human AML (Fig. A-B). This reinforces the hypothesis that aberrant PDPN expression may be a strong and unappreciated contributor to platelet consumption in APL. Conclusion:I) CEPBE is recurrently mutated in a small subset of APL patients. II) PDPN is the most specific aberrant transcript in this disease and is a new biomarker for APL. Systematic assessment of podoplanin by flow cytometry in newly diagnosed AML could lead to an earlier detection of unsuspected APL cases. III) PDPN expression by leukemic promyelocytes likely contributes to defective primary hemostasis, representing a new mechanism of APL-related bleeding. This provides a strong rationale for evaluating PDPN-CLEC-2 axis inhibitors in this setting. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Scl operates both downstream of Kit to control the survival of Kit+ multipotent and erythroid progenitors and upstream of Kit to determine Kit expression levels. Scl and Kit establish a positive feedback loop in hematopoietic progenitors.

Description: RUNX1 is an essential transcription factor for definite hematopoiesis and plays important roles in immune function. Mutations in RUNX1 occur in 5-13% of Acute Myeloid Leukemia (AML) patients (RUNX1mut ) and are associated with adverse outcome, highlighting the need for better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for patients with RUNX1mut AML. Toward this goal, we performed RNA-sequencing of a cohort of RUNX1mut primary AML specimens and used a chemogenomic approach combining mutational and gene expression analysis of these specimens with assessment of their drug sensitivity profile through chemical screening. Sequencing data analysis demonstrated that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Interestingly, these studies also unveiled the increased susceptibility of RUNX1-mutated specimens to Glucocorticoids (GCs) and revealed that RUNX1 allele dosage dictates sensitivity to these compounds in AML patient cells, unravelling a new role for RUNX1 in the Glucocorticoid Receptor (GR) pathway. GR is a nuclear receptor that modulates the expression of thousands of genes involved in several biological processes such as metabolism, immune function, skeletal growth, etc. GCs are commonly used to treat cancers of the lymphoid system, however their potential benefits for AML treatment have never been assessed formally and the mechanism of action of these drugs is not fully understood. Transcriptome analyses identified NR3C1 (encoding the GR) as one of the genes whose expression is determined by RUNX1 allele dosage, with increased expression in RUNX1mut specimens, indicating that RUNX1 inactivation could lead to GR upregulation, which might explain the increased sensitivity to GCs. We previously showed that RUNX1 silencing sensitizes human AML cell lines to GCs and that this acquired sensitivity is accompanied by the upregulation of the GR both at the transcript and protein levels. However, basal levels of GR could not explain GC sensitivity in all cases, indicating that other mechanisms are involved in the GC response. By performing co-immunoprecipitations (co-IP), we demonstrated that RUNX1 and GR physically interact in AML cells. Overexpression of FLAG-tagged RUNX1 mutants in HEK293 cells followed by co-IP identified the C-terminal inhibitory domain of RUNX1 as essential for the interaction with GR. Our results suggest that RUNX1 could be part of the GR transcriptional complex and could modulate the transcription of genes involved in the response to GCs. To identify regulators of the GC response, we are currently performing genome-wide CRISPR-Cas9-based genetic screens in AML cell lines. We generated RUNX1-deficient (GCs sensitive) AML cell lines that uniformly express Cas9 under a doxycycline-inducible promoter. These cell lines, along with parental cell lines (GC resistant) were used to conduct screens in GCs-supplemented media for the identification of genes that confer sensitivity or resistance to GCs. Mechanistic insights gained from these experiments will allow the design of additional therapeutic strategies to potentiate the effect of GCs on poor outcome AML. Disclosures No relevant conflicts of interest to declare.

Description: Key Points CSF3R was the most frequently mutated gene identified in this CEBPAbi AML cohort analyzed by next-generation sequencing. CEBPA bi AML that have a characteristic transcriptomic profile are more sensitive to JAK inhibitors than CEBPAwt AML.

Description: Acute myeloid leukemia (AML) is associated with poor overall survival and the development of more effective therapies is urgently needed. G Protein-Coupled Receptors (GPCRs) represent the largest family of membrane receptors, with an estimated 800 members in humans, and are attractive therapeutic targets, accounting for approximately 30% of targets of marketed drugs. These receptors are key transducers that bind a vast diversity of ligands (e.g. glycoproteins, peptides, amino acids, nucleotides, nucleosides, ions) allowing the cells to adapt to their environment by regulating a wide variety of physiological processes including the control of blood pressure, heart rate, digestive processes, hormone secretion, cell growth and migration, as well as vision and olfaction. Binding to their ligands leads to conformational rearrangements promoting the engagement and modulation of many distinct downstream signaling effectors that are both G protein-dependent and independent. Several GPCRs are critical for cell proliferation and survival, and can be aberrantly expressed in cancer cells. For example, the chemokine receptor CXCR4 plays an important role in metastasis and angiogenesis in breast cancer and many other types of tumors. In AML, CXCR4 overexpression has been associated with poor outcome. Moreover, in vivo mouse studies have shown that the use of a small molecule antagonist of CXCR4 increases the mobilization of AML cells into the peripheral blood and improves the apoptotic effects of chemotherapy. This activity has been explored in a phase I/II clinical study showing that the addition of CXCR4 antagonists to chemotherapy in AML might improve the remission rate. The role of GPCRs in mouse leukemogenesis has also been suggested in a transcriptome analysis of two related leukemia clones that differ in their stem cell frequency. In this study, GPCRs were the most differentially expressed class of genes between the two clones. Currently, an extensive assessment of GPCR expression in human AML is lacking. To address this issue, we studied the expression of GPCRs in a large cohort of AML samples, as well as in normal blood cells, bone marrow cell populations, and cord blood-derived CD34+ cells as controls.The 772 GPCRs analyzed in this study consist of all the GPCR members included in the International Union of Basic and Clinical Pharmacology (IUPHAR) database, as well as 370 olfactory, 24 taste and 4 vomeronasal receptors. RNA sequencing data analysis was performed as previously described (Lavallée et al, Blood 2015 Jan 1;125(1):140-3) and revealed that 240 GPCRs are expressed in cells from this AML cohort. Among these receptors, 30 are upregulated and 19 are downregulated in AML samples compared to CD34+ normal cells. Upregulated GPCRs are enriched in chemokine (CCR1, CXCR4, CCR2, CX3CR1, CCR7, and CCRL2), adhesion (CD97, EMR1, EMR2, and GPR114) and purine (including P2RY2 and P2RY13) receptor sub-families. The downregulated members include adhesion GPCRs such as LPHN1, GPR125, GPR56, CELSR3, and GPR126, protease-activated receptors (F2R and F2RL1), and the Frizzled family receptors SMO and FZD6. Interestingly, specific deregulation was observed in genetically distinct subgroups of AML, a subset of GPCRs being differentially expressed in normal karyotype AML with NPM1 or FLT3 -ITD mutations, and in specimens with Core Binding Factor and MLL rearrangements, thereby representing promising therapeutic targets or diagnostic markers. In conclusion, our results demonstrate that several GPCR members are deregulated in AML with a clear enrichment in distinct classes, providing the rationale for functional assays using available agonists or antagonists to leukemia-enriched GPCRs. Since these receptors are the targets of several US Federal and Drug Administration approved drugs, our results pave the way to explore selected GPCRs as novel AML therapeutic targets. Disclosures Bouvier: American Society of Nephrology: Other: speaker; Domain Therapeutics: Other: Company SAB member, Research Funding; Vertex Pharmaceutical: Research Funding; Ontario Genomic Institute: Other: SAB meeting; BMS: Research Funding; DalCor Pharmaceutics: Other: Company SAB member; Pfizer: Other: Speaker, Research Funding; Novo-Nordisc: Research Funding.

Description: The SCL and LMO oncogenes are frequently activated in childhood T cell acute leukemia (T-ALL). SCL is a bHLH transcription factor that forms heterodimers with other members, specifically HEB and E2A. SCL can either activate or repress transcription but its mechanism of action as an oncogene remains to be clarified. Ectopic expression of SCL and LMO in transgenic mice causes thymocyte differentiation arrest during the preleukemic phase with aberrant differentiation at the DN3–DN4 stage, prior to the acquisition of CD4 and CD8. We therefore took several approaches to define the mechanism underlying this differentiation arrest. Using a CD3e-deficient background to eliminate preTCR signaling effects, an event that is not essential for leukemogenesis, we first analyzed global gene expression of pre-leukemic SCLtg/LMOtg DN3 thymocytes against their non-transgenic littermates via the Affymetrix platform. In this context, SCL/LMO globally affects the expression of both positive and negative targets of E2A/HEB. These results were confirmed by real-time quantitative PCR, showing that SCL/LMO repress E2A/HEB activity. Second, bioinformatic analysis of genes significantly affected by SCL/LMO allowed us to identify phylogenetically conserved HEB/E2A binding sites within the promoter regions of half of these genes. E2A/HEB binding was directly assessed through chromatin immunoprecipitation of primary thymocytes, confirming known target genes and revealing novel ones. This assay also showed that SCL associates with HEB and/or E2A on DNA. Moreover, structure function analysis indicate that transcription inhibition by SCL depends on its HLH domain but not on LMO interaction. We therefore conclude that SCL inhibits thymocyte differentiation by inhibiting E2A/HEB targets that include genes required for thymocyte differentiation, cell signalling, DNA repair and replication. Finally, using a genetic approach we show that SCL/LMO collaborates with HEB haploinsufficiency in inducing leukemia. Our observations therefore reveal that the repression of HEB/E2A by SCL/LMO is a crucial step in T cell transformation. Support from CIHR and FRSQ.