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Key Points Inhibition of RAB protein function mediates the anti–acute myeloid leukemia activity of statins.Statin sensitivity is associated with enhanced vesicle-mediated traffic., Visual Abstract, Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.

RUNX1 is mutated in ∼10% of adult acute myeloid leukemia (AML). Although most RUNX1 mutations in this disease are believed to be acquired, they can also be germline. Indeed, germline RUNX1 mutations result in the well-described autosomal-dominant familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD, FPD/AML, FPDMM); ∼44% of affected individuals progress to AML or myelodysplastic syndromes. Using the Leucegene RUNX1 AML patient group, we sought to investigate the proportion of germline vs acquired RUNX1 mutations in this cohort. Our results showed that 30% of RUNX1 mutations in our AML cohort are germline. Molecular profiling revealed higher frequencies of NRAS mutations and other mutations known to activate various signaling pathways in these patients with RUNX1 germline–mutated AML. Moreover, 2 patients (mother and son) had co-occurrence of RUNX1 and CEBPA germline mutations, with variable AML disease onset at 59 and 27 years, respectively. Together, these data suggest a higher than anticipated frequency of germline RUNX1 mutations in the Leucegene cohort and further highlight the importance of testing for RUNX1 mutations in instances in which allogeneic stem cell transplantation using a related donor is envisioned.

BackgroundThe stimulation and expansion of antigen-specific T cells ex vivo enables the targeting of a multitude of cancer antigens. However, clinical scale T-cell expansion from rare precursors requires repeated stimulations ex vivo leading to T-cell terminal effector differentiation and exhaustion that adversely impact therapeutic potential. We leveraged immune checkpoint blockade relevant to antigen-specific CD8+ human T cells to improve the expansion and function of T cells targeting clinically relevant antigens.MethodsA clinically-compliant protocol relying on peptide-pulsed monocyte-derived dendritic cells and cytokines was used to expand antigen-specific CD8+ targeting the oncogenic Epstein-Barr virus (EBV) and the tumor associated antigen (TAA) Wilms Tumor 1 (WT1) protein. The effects of antibody-mediated blockade of immune checkpoints applied to the cultures (T-cell expansion, phenotypes and function) were assessed at various time points. Genomic studies including single cell RNA (scRNA) sequencing and T-cell receptor sequencing were performed on EBV-specific T cells to inform about the impact of immune checkpoint blockade on the clonal distribution and gene expression of the expanded T cells.ResultsSeveral immune checkpoints were expressed early by ex vivo expanded antigen-specific CD8+ T cells, including PD-1 and TIM-3 with co-expression matching evidence of T-cell dysfunction as the cultures progressed. The introduction of anti-PD-L1 (expressed by the dendritic cells) and anti-TIM-3 antibodies in combination (but not individually) to the culture led to markedly improved antigen-specific T-cell expansion based on cell counts, fluorescent multimer staining and functional tests. This was not associated with evidence of T-cell dysfunction when compared to T cells expanded without immune checkpoint blockade. Genomics studies largely confirmed these results, showing that double blockade does not impart specific transcriptional programs or patterns on TCR repertoires. However, our data indicate that combined blockade may nonetheless alter gene expression in a minority of clonotypes and have donor-specific impacts.ConclusionsThe manufacturing of antigen-specific CD8+ T cells can be improved in terms of yield and functionality using blockade of TIM-3 and the PD-L1/PD-1 axis in combination. Overcoming the deleterious effects of multiple antigenic stimulations through PD-L1/TIM-3 blockade is a readily applicable approach for several adoptive-immunotherapy strategies.

Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy-resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome RNA and miRNA sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of apparent NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.

In acute myeloid leukemia (AML), chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL–AF9. MEF2C phosphorylation was required for leukemia stem cell maintenance and induced by MARK kinases in cells. Treatment with the selective MARK/SIK inhibitor MRT199665 caused apoptosis and conferred chemosensitivity in MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease. Significance: Functional proteomics identifies phosphorylation of MEF2C in the majority of primary chemotherapy-resistant AML. Kinase-dependent dysregulation of this transcription factor confers susceptibility to MARK/SIK kinase inhibition in preclinical models, substantiating its clinical investigation for improved diagnosis and therapy of AML. Cancer Discov; 8(4); 478–97. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 371

Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.

Purpose: RUNX1-mutated (RUNX1mut) acute myeloid leukemia (AML) is associated with adverse outcome, highlighting the urgent need for a better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for this disease. Toward this goal, we further dissected the mutational spectrum and gene expression profile of RUNX1mut AML and correlated these results to drug sensitivity to identify novel compounds targeting this AML subgroup. Experimental Design: RNA-sequencing of 47 RUNX1mut primary AML specimens was performed and sequencing results were compared to those of RUNX1 wild-type samples. Chemical screens were also conducted using RUNX1mut specimens to identify compounds selectively affecting the viability of RUNX1mut AML. Results: We show that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Chemical screening revealed that most RUNX1mut specimens are sensitive to glucocorticoids (GCs) and we confirmed that GCs inhibit AML cell proliferation through their interaction with the glucocorticoid receptor (GR). We observed that specimens harboring RUNX1 mutations expected to result in low residual RUNX1 activity are most sensitive to GCs, and that coassociating mutations as well as GR levels contribute to GC sensitivity. Accordingly, acquired glucocorticoid sensitivity was achieved by negatively regulating RUNX1 expression in human AML cells. Conclusions: Our findings show the profound impact of RUNX1 allele dosage on gene expression profile and glucocorticoid sensitivity in AML, thereby opening opportunities for preclinical testing which may lead to drug repurposing and improved disease characterization. Clin Cancer Res; 23(22); 6969–81. ©2017 AACR.

Compared to solid tumors, antibody-based immunotherapy is underexploited in acute myeloid leukemia (AML), likely due to the limited availability of targetable surface antigens specifically expressed on AML cells. While targeted immunotherapies hold great promise, their development inexorably depends on the identification of cell surface antigens selectively expressed by cancer cells. The identification of such antigens has been challenging due to the characteristic hydrophobicity of plasma membrane proteins. Another level of complexity is brought by the fact that AML is a heterogeneous disease comprised of different subgroups with associated prognosis ranging from favorable to adverse. In order to identify novel AML antigens to be targeted using immunotherapeutic approaches, we have analysed the surface proteome of a collection of 100 primary human AML specimens, enriched in poor prognosis AML samples. These analyses revealed that AML subgroups have distinct surfaceome signatures. We also confirmed the expression of known AML markers at the surface of specific AML subgroups. Our search for novel AML antigens focused on four poor prognosis AML subgroups: MLL-rearranged, complex karyotype, RUNX1-mutated and normal karyotype with NPM1, DNMT3A and FLT3 (ITD) mutations. We identified proteins selectively expressed at the surface of AML cells from these subgroups. Analysis of the whole cohort of specimens also identified known and novel pan AML antigens. Single cell RNA-sequencing analysis of aforementioned subgroup-specific and pan AML antigens revealed that many of these are expressed by the vast majority of blast populations within primary AML specimens, including most primitive ones. Overall, these results suggest that studying the surface proteome of AML cells has the potential to reveal promising AML antigens to be targeted using immunotherapeutic approaches.

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis. Single-cell analysis of lung cancer progression uncovers developmental and regenerative programs co-opted by cancer cells and immune-mediated pruning during metastatic outbreak

Acute myeloid leukemias (AML) with mutations in the NPM1 gene (NPM1c+) represent a large AML subgroup with varying response to conventional treatment, highlighting the need to develop targeted therapeutic strategies for this disease. We screened a library of clinical drugs on a cohort of primary human AML specimens and identified the BCL2 inhibitor ABT-199 as a selective agent against NPM1c+ AML. Mutational analysis of ABT-199-sensitive and -resistant specimens identified mutations in NPM1, RAD21, and IDH1/IDH2 as predictors of ABT-199 sensitivity. Comparative transcriptome analysis further uncovered BCL2A1 as a potential mediator of ABT-199 resistance in AML. In line with our observation that RAD21 mutation confers sensitivity to ABT-199, we provide functional evidence that reducing RAD21 levels can sensitize AML cells to BCL2 inhibition. Moreover, we demonstrate that ABT-199 is able to produce selective anti-AML activity in vivo toward AML with mutations associated with compound sensitivity in PDX models. Overall, this study delineates the contribution of several genetic events to the response to ABT-199 and provides a rationale for the development of targeted therapies for NPM1c+ AML.

Patients diagnosed with acute myeloid leukemia with complex karyotype (CK AML) have an adverse prognosis using current therapies, especially when accompanied by TP53 alterations. We hereby report the RNA-sequencing analysis of the 68 CK AML samples included in the Leucegene 415 patient cohort. We confirm the frequent occurrence of TP53 alterations in this subgroup and further characterize the allele expression profile and transcript alterations of this gene. We also document that the RAS pathway (N/KRAS, NF1, PTPN11, BRAF) is frequently altered in this disease. Targeted chemical interrogation of genetically characterized primary CK AML samples identifies polo-like kinase 1 (PLK1) inhibitors as the most selective agents for this disease subgroup. TP53 status did not alter sensitivity to PLK1 inhibitors. Interestingly, CK AML specimens display a G2/M transcriptomic signature that includes higher expression levels of PLK1 and correlates with PLK1 inhibition sensitivity. Together, our results highlight vulnerability in CK AML. In line with these in vitro data, volasertib shows a strong anti-AML activity in xenotransplantation mouse models of human adverse AML. Considering that PLK1 inhibitors are currently being investigated clinically in AML and myelodysplastic syndromes, our results provide a new rationale for PLK1-directed therapy in patients with adverse cytogenetic AML.

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.

In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.

Identification of MYC mutations in acute myeloid leukemias with NUP98 – NSD1 translocations

Complex karyotype (CK) acute myeloid leukemia (AML), especially those with TP53 anomalies, are particularly resistant to current therapies and remain one of the most heterogeneous and poorly characterized genetic group. Using global transcriptomic analyses, we identified the oncofetal HMGA2 gene as one of the most upregulated gene in CK AML. HMGA2 is a known stem cell gene and we showed, at single cell level, that HMGA2 is indeed expressed in normal hematopoietic stem cells (HSCs) but also in primitive CD34+ leukemic cells. Abnormal re-expression of HMGA2 has been reported in several tumor types and linked to chemo-resistance, advanced tumor grade and poor prognosis. Using a CRISPR/Cas9 genomic screening approach, we identified vulnerabilities in leukemic cells overexpressing HMGA2. Among them, we found that targeting the G2/M cell cycle phase transition was deleterious to those cells. We confirmed these observations using chemical inhibitors of the G2/M regulators ATR, CHK1 and WEE1 and found that these compounds were preferentially active on HMGA2+ CK AML specimens. Our findings thus suggest that HMGA2 is a key functional determinant in AML associated with stem cell features, G2/M perturbation and related drug sensitivity.

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.

Survival for children with acute myeloid leukemia (AML) remains low, with a primary induction remission failure rate of 10-15%. Although some mutations have been identified that are associated with increased relapse risk, the genomic landscape of pediatric AML resistant to primary chemotherapy is not well characterized. As part of the TARGET initiative (Therapeutically Applicable Research To Generate Effective Treatments), we assembled a cohort of 28 pediatric AML patients with primary chemotherapy resistance and failure of induction chemotherapy, uniformly treated as part of the COG AAML0531 study. We analyzed whole-genome DNA, mRNA, and miRNA sequence data obtained at diagnosis and after induction chemotherapy. At least three genetically distinct groups were apparent in this cohort, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of NUP98 variants, and additional recurrent variants including those in KMT2C and MLLT10. Comparisons of mutations before and after chemotherapy revealed both common and distinct gene expression programs. On chemotherapy exposure, these leukemias exhibited diverse forms of clonal evolution. We observed selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination or contraction of FLT3, PTPN11, and NRAS mutant clones. These findings suggest diverse genetic mechanisms for primary chemotherapy resistance in pediatric AML, which should guide new approaches for its diagnosis and therapy. Citation Format: Nicole McNeer, John Philip, Heather Geiger, Rhonda E. Ries, Vincent-Philippe Lavallee, Michael Walsh, Minita Shah, Kanika Arora, Anne-Katrin Emde, Nicolas Robine, Todd A. Alonzo, E. Anders Kolb, Alan S. Gamis, Malcom Smith, Daniela Se Gerhard, Jaime Guidry-Auvil, Soheil Meshinchi, Alex Kentsis. Genetic mechanisms of primary chemotherapy resistance in pediatric acute myeloid leukemia [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 2870.

Appropriate culture methods for the interrogation of primary leukemic samples were hitherto lacking and current assays for compound screening are not adapted for large-scale investigation of synergistic combinations. In this study, we report a novel approach that efficiently distills synthetic lethal interactions between small molecules active on primary human acute myeloid leukemia (AML) specimens. In single-dose experiments and under culture conditions preserving leukemia stem cell activity, our strategy considerably reduces the number of tests needed for the identification of promising compound combinations. Initially conducted with a selected library of 5000 small molecules and 20 primary AML specimens, it reveals 5 broad classes of sensitized therapeutic target pathways along with their synergistic patient-specific fingerprints. This novel method opens new avenues for the development of AML personalized therapeutics and may be generalized to other tumor types, for which in vitro cancer stem cell cultures have been developed.

Based on transcriptomic analyses of thousands of samples from The Cancer Genome Atlas, we report that expression of constitutive proteasome (CP) genes (PSMB5, PSMB6, PSMB7) and immunoproteasome (IP) genes (PSMB8, PSMB9, PSMB10) is increased in most cancer types. In breast cancer, expression of IP genes was determined by the abundance of tumor infiltrating lymphocytes and high expression of IP genes was associated with longer survival. In contrast, IP upregulation in acute myeloid leukemia (AML) was a cell-intrinsic feature that was not associated with longer survival. Expression of IP genes in AML was IFN-independent, correlated with the methylation status of IP genes, and was particularly high in AML with an M5 phenotype and/or MLL rearrangement. Notably, PSMB8 inhibition led to accumulation of polyubiquitinated proteins and cell death in IPhigh but not IPlow AML cells. Co-clustering analysis revealed that genes correlated with IP subunits in non-M5 AMLs were primarily implicated in immune processes. However, in M5 AML, IP genes were primarily co-regulated with genes involved in cell metabolism and proliferation, mitochondrial activity and stress responses. We conclude that M5 AML cells can upregulate IP genes in a cell-intrinsic manner in order to resist cell stress.

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.

Background: Acute myeloid leukemia (AML) with complex karyotype (CK) is associated with adverse prognosis with current therapies, in particular in the presence of TP53 mutations. Identification of novel therapeutic strategies is therefore urgently needed for this subgroup of patients. Methods and aims: As part of the Leucegene project, we RNA sequenced a cohort of 415 clinically annotated primary human AML specimens. Comparative transcriptomic analysis of the 68 CK AML specimens with the non-CK samples of the cohort was performed to identify features associated with the CK AML subgroup. Based on these results, we performed a chemical screen aimed at identifying active agents toward CK AML. Gene expression analysis, primary AML specimen culture and chemical screening were performed as previously described by our group (Lavallée et al., Nature Genetics, 2015; Pabst et al., Nature Methods, 2014; Simon et al., Clinical cancer research, 2017). Results: CK AML is a heterogeneous disease by definition and accordingly, Multidimensional Scaling (MDS) analysis fails to identify a distinct transcriptional signature. Nonetheless, comparative transcriptome analysis of CK and non-CK AML specimens revealed highly deregulated genes between these subgroups (Figure 1A). Among them, EDA2R, a known target to p53, is specifically downregulated in CK AML. This reflects the frequent TP53 alterations observed in CK AML. Most importantly, we found that the recently identified AML prognostic gene HMGA2 is overexpressed in the majority of these patients, independently of the TP53 status. HMGA2 is an oncofetal gene expressed in hematopoietic stem cell (HSC) -enriched populations while its expression decreases in progenitors and is nearly absent in mature cells. Interestingly, Gene Set Enrichment Analysis (GSEA) revealed an enrichment for HSC genes in AML specimens with high HMGA2 expression levels, which could reflect a stem-cell origin or a more immature state for these leukemias. Global transcriptomic analysis of CK AML also identified a G2/M checkpoint signature for HMGA2-high specimens (Figure 1B). Accordingly, we observed that chemical inhibitors of the G2/M regulators ATR, CHK1 and WEE1 are preferentially active on HMGA2-high CK specimens and that engineered overexpression of HMGA2 in different leukemia cell lines enhanced sensitivity to G2/M inhibition (Figure 1C). This specific inhibition was independent of the TP53 status. Conclusions: Here we report a comprehensive transcriptomic analysis of the CK AML specimens of the Leucegene cohort and identified aberrant expression of the HMGA2 oncogene in close to 80% of these samples. Chemical interrogation of primary AML specimens revealed a TP53-independent but HMGA2-mediated sensitization of CK AML to G2/M checkpoint inhibition. Thus, our results reveal a novel vulnerability for AML expressing high levels of HMGA2 and identify potential biological targets for these high-risk patients. Since several CHK1 and PLK1 inhibitors have been or are currently under evaluation in clinical trials, our findings suggest that HMGA2-high AML patients could benefit from this therapeutic approach. Figure 1: A/ Volcano plot representing the genes differentially expressed in CK versus non-CK AML. Samples with low group median expression (2; n=39) and HMGA2 null (RPKM=0; n=83) AML specimens. C/ Dose-response curves and IC50 values for representative compounds in K562 cells infected with control YFP vector (blue) or HMGA2-YFP expressing vector (red). Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

Acute myeloid leukemias (AML) are aggressive blood cancers characterized by an overall survival of 27% at 5 years. The main challenge in AML treatment originates from the genetic heterogeneity of the disease that contributes to the wide range of clinical outcomes observed. A large proportion (~35%) of AML patients exhibit no distinguishable chromosomal abnormalities that can be used to guide treatment selection and are therefore classified in the poorly characterized intermediate risk category. Approximately 50-60% of intermediate risk AML patients carry mutations in the NPM1 gene. These mutations are associated with a favorable outcome unless a concomitant mutation in the FLT3 gene is detected, which accounts for 39% of cases. The survival rate is further worsened when a third mutation is detected in the DNMT3A gene, dropping from 40% to 20% 5 years post-treatment for double and triple mutants, respectively. This study aimed to identify drugs selectively affecting the viability of leukemic cells from AML patients with NPM1 mutations. To achieve this, we took advantage of culture conditions developed by our group that prevent differentiation of leukemic cells and preserve leukemia stem cell activity from primary AML specimens (Pabst et al., Nature methods, 2014), enabling chemical screening of primary AML specimens. We conducted a chemical screen using a collection of ~300 clinical grade drugs on a cohort of 38 primary human AML specimens containing NPM1 mutated (NPM1c+) and NPM1 wild-type (NPM1wt) samples. These specimens belonged to the Leucegene collection of sequenced and clinically annotated samples. The screen identified ABT-199 as the compound with the most discriminatory effect toward NPM1c+ AML. ABT-199 is a specific BH3-mimetic that prevents anti-apoptotic BCL2 from binding pro-apoptotic BAX and BAK1 proteins, leading to apoptosis. ABT-199 demonstrated encouraging results for AML treatment, but the determinants of drug sensitivity have not been well defined. Analysis of the enrichment of clinical variables in ABT-199 sensitive and resistant groups of AML specimens to define characteristics/biomarkers associated with ABT-199 sensitivity in AML revealed that mutations in NPM1, RAD21, IDH1, IDH2, DNMT3A and FLT3 (ITD), as well as normal karyotype and the FAB M1 class all significantly associate with increased ABT-199 sensitivity. At the other side of the spectrum, mutations in TP53 and FAB class M5B were significantly enriched in the resistant group. Additional analyses revealed that NPM1c+/DNMT3Amut/FLT3-ITD specimens are sensitive to ABT-199, which may provide a rationale to prioritize patients from this adverse risk AML subgroup for explorative ABT-199 based regimens. Specimens with RAD21 mutations were the most sensitive to ABT-199 treatment and further analyses demonstrated a clear association between mutation of cohesin genes (RAD21, SMC1A, SMC3, STAG2) and increased ABT-199 sensitivity. In line with this, we demonstrated that RAD21 knockdown alone is able to sensitise AML cell lines to BCL2 inhibition. Comparative transcriptome analysis of ABT-199 sensitive and resistant specimens also revealed an apoptotic gene signature linked to ABT-199 resistance with BCL2A1, an anti-apoptotic BCL2 homolog, being the most differentially expressed apoptotic gene between these response groups and showing increased expression in the resistant subset. Expression correlation analysis over the 415 specimens of the Leucegene cohort showed that BCL2A1 is one of the top genes anti-correlated to BCL2, and accordingly, high BCL2 and BCL2A1 expressors were enriched among ABT-199 sensitive and resistant specimens, respectively. In conclusion, using an unbiased pharmacogenomic approach, we identified ABT-199, a compound with the potential to eradicate NPM1c+ AML, which has already been tested in a phase 2 clinical trial for AML. Our results shed light on determinants of ABT-199 sensitivity which could readily impact AML therapy by providing a rationale for prioritizing patients with NPM1, RAD21, IDH1 and/or IDH2 mutations for ABT-199 AML trials. Our results also uncover potential mechanisms of resistance to ABT-199, providing grounds to design combination therapies to overcome ABT-199 chemoresistance. Disclosures Sauvageau: ExCellThera: Employment, Equity Ownership.

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.

Background: Acute myeloid leukemias (AML) with complex karyotype (CK) are associated with an adverse patient prognosis with current therapies, in particular when they include TP53 mutations. Identification of novel therapeutic strategies is urgently needed for this subgroup of patients. Methods and aims:We have performed an RNA-Sequencing transcriptomic analysis of 68 CK AML included in the Leucegene cohort comprising 415 primary AML samples and performed correlative targeted chemical screening aiming at the identification of active agents in this subgroup. Cell culture and analysis of mutations, gene expression and chemical screening were performed as previously described (Pabst et al., Nature Methods, 2014; Lavallée et al, Nature Genetics, 2015). Results:Genes mutated at a frequency greater than 5% in the CK cohort were: TP53 (43/68, 63%), NRAS (9/68, 13%), DNMT3A (8/68, 12%), TET2 and NF1 (7/68 each, 10%), RUNX1 (6/68), FLT3, MLL and PTPN11 (5/68 each) and JAK2 and U2AF1 (4/68 each). Of those, only TP53 (p < 0.0001) and NF1 (p = 0.02) were preferentially associated with CK AML. HMGA2 (High Mobility Group AT-hook 2) was the most differentially expressed gene in CK AML compared to other AML (median: 1.35 vs 0.008 RPKM, q = 2 x 10-14). TP53-mutated samples were in addition characterized by low expression levels of Ectodysplasin A2 Receptor or EDA2R, a known target of TP53. We next tested in dose-response studies 267 compounds enriched in approved anti-cancer drugs on 27 primary CK AML samples and in 11 intermediate-risk karyotype controls. Four percent of these compounds were very active (median IC50 < 10 nM) in CK AML (red dots in Fig A). Surprisingly, this small subset of compounds comprised the only 2 inhibitors of Polo-Like Kinase 1 (PLK1), volasertib (median IC50: 6.6 nM) and rigosertib (median IC50: 8.5 nM). Rigosertib was, in addition, more active in CK AML than in intermediate-risk AML (p = 0.0035) supporting the hypothesis that choice of treatment, rather than genetic anomalies, determine prognosis (Fig. B-C). Most interestingly, expression levels of PLK1 correlated with the measured sensitivity to both rigosertib and volasertib raising the possibility that PLK1 expression determines sensitivity to these inhibitors. PLK1 is more highly expressed in CK AML (p=0.009) and in TP53-mutated AML (p = 0.015) than in control samples. A threshold of 5 RPKM was particularly predictive for drug sensitivity (rigosertib: p < 0.001, Fig C; volasertib: p = 0.037) suggesting that it could become a companion biomarker. Rigosertib and volasertib also potently inhibit PLK2 and PLK3 in cell-free assays, but no association was observed between PLK2/PLK3 expression and response to inhibitors, suggesting that cell lethality was mediated by PLK1 inhibition. PLK1 is a protein kinase involved in cell cycle. Accordingly, expression of this gene in our dataset defines a cluster of very highly correlated genes comprising mostly cell cycle components (e.g. CDC20, CCNB2, CENPA and CTSE1: > 0.90). Volasertib was recently studied in a phase 2 clinical trial in AML patients in combination with low-dose cytarabine (LDAC) (Döhner et al, Blood 2014). In this analysis, and in line with our data, responses were seen across all genetic groups including in adverse-risk AML (1/14 in LDAC vs 5/14 in LDAC + volasertib). Conclusion:Our chemo-transcriptomic analysis revealed that CK AML are characterized by high levels of HMGA2 expression and, in addition for the TP53 mutated subset, low levels of EDA2R. Most importantly our results show that CK AML with high expression levels of PLK1 are uniformly and preferentially sensitive to PLK1 inhibitors. Our data thus support the hypothesis that sensitivity to PLK1 inhibitors is associated to PLK1 expression, and that this gene may represent a promising biomarker to predict biologicalresponse to these agents. Our analysis provides for the first time a strong rationale for investigating PLK1 inhibitors in the context of CK and/or TP53 mutated AML in which correlative PLK1 expression analyses are commanded. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is associated with poor clinical outcome and the development of more effective therapies is urgently needed. G protein-coupled receptors (GPCRs) represent attractive therapeutic targets, accounting for approximately 30% of all targets of marketed drugs. Using next-generation sequencing, we studied the expression of 772 GPCRs in 148 genetically diverse AML specimens, normal blood and bone marrow cell populations as well as cord blood-derived CD34-positive cells. Among these receptors, 30 are overexpressed and 19 are downregulated in AML samples compared with normal CD34-positive 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 subfamilies. The downregulated receptors 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, thereby identifying different potential therapeutic targets in these frequent AML subgroups.

Insights into the complex clonal architecture of acute myeloid leukemia (AML) unravelled by deep sequencing technologies have challenged the concept of AML as a hierarchically organised disease initiated and driven by rare self-renewing leukemic stem cells (LSCs). In contrast to normal human hematopoietic stem cells (HSCs), which are highly enriched in the CD34+ CD38- population, LSCs have also been found in the CD34- and the CD38+ fractions questioning the existence of a consistent LSC surface marker profile for AML. Besides, low LSC frequencies in primary samples, rapid onset of differentiation upon ex vivo culture, and genetic inter-specimen heterogeneity hamper the dissection of the molecular machinery that drives LSC self-renewal. We performed RNA-Sequencing of primary human AML samples and assessed LSC frequencies by limiting dilution analyses for 56 of these in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. By comparing gene expression profiles between high vs low LSC frequency leukemias, we identified the G-protein coupled receptor 56 (GPR56) has significantly more expressed in high LSC frequency leukemias. We validated the RNA-seq data with protein expression by FACS and found an excellent correlation. To determine whether GPR56 positive cells overlapped with the known LSC-associated phenotype CD34+ CD38-, we stained 45 AML samples with CD34, CD38, GPR56, and antibodies against other described LSC markers. Although CD34+ GPR56+ and CD34+ CD38- compartments identified the same population in some samples, we found in the majority of samples that GPR56 further subdivided the CD34+ CD38- compartment. Accordingly, not only the proportions of total GPR56+ and CD34+ GPR56+ cells were significantly higher in LSChigh versus LSClow samples, but also the proportion of GPR56+ cells within the CD34+ CD38- compartment was significantly different between the groups indicating that GPR56 might be of additional value to what is currently considered the best described LSC phenotype. The percentage of total CD34 positive cells did not correlate with LSC frequency clearly distinguishing GPR56 from CD34 or CD38, which are only suitable LSC markers when used in combination. We analysed other potential LSC markers (TIM3, CD96, CD44, CD123, CLL1 and CD47) in our RNA-Seq dataset and by FACS analysis in combination with CD34 as we did for GPR56 and none of them correlated with LSC frequency in our sample collection. To determine whether GPR56 discriminates engrafting LSCs from non-LSCs, we sorted GPR56+ and GPR56- cells within the CD34-positive and -negative compartments from selected specimens with known engraftment potential. We found that GPR56 identified the engrafting fraction in CD34positive AML samples, with a >50 fold enrichment in LSC in the CD34+GRP56+ fraction vs the CD34+GPR56- fraction within the same sample, demonstrating that GPR56 is a good LSC marker. Specimens with high molecular or cytogenetic risk such as chromosome 5 or 7 anomalies and EVI1- rearrangementexpressed high levels of both, GPR56 and CD34, while samples with coexistent FLT3 -ITD, DNMT3A, and NPM1 mutations displayed a unique CD34low GPR56high profile. Moreover, we found a divergent distribution of variant allele frequencies in GPR56+ versus GPR56- fractions identifying GPR56 as a discriminator of leukemic sub-clones with high and low NSG engrafting capacity. Analysis of engrafted cells re-sorted based on GPR56 after being harvested from mouse bone marrow revealed reduced complexity of the clonal composition. Most importantly, GPR56 positive cells differentiated to GPR56 negative cells in mice, which did not happen in the human niche, in which GPR56 positive and negative fractions represented two independently evolved subclones. In summary our work identifies GPR56 as a novel LSC marker in AML and also shows that GPR56 readily identifies a functionally distinct LSC-rich subclone in the majority of human AML patients and reveals hitherto unforeseen complexity in the interaction between human LSCs and the NSG mouse environment. Disclosures No relevant conflicts of interest to declare.

Solid tumors at the advanced stages are often characterized by the overexpression of tyrosine kinase receptors that stimulate growth through the MAP kinase pathway and activate antiapoptotic signaling through the PI3K kinase pathway. One such receptor is the epidermal growth factor receptor (EGFR) that is overexpressed in many tumors including brain, lung, breast, ovarian and prostate carcinomas. EGFR can synergize with other tyrosine kinases such as Src to promote invasion and metastasis. However, despite the implication of EGFR in multiple processes, inhibitors of its tyrosine kinase activity such as ZD1839 or erlotinib showed moderate antiproliferative activity against certain tumor types in the clinic. It has been reported that EGFR expressing cells that harbor dysfunctional PTEN, are moderately sensitive to EGFR inhibitors. Given the complexity of cell response the latter class of inhibitors, we thought it of interest to investigate agents that are not only directed at EGFR but also to divergent targets such as Src or DNA, with the purpose of producing single compounds with greater potency than their single inhibitor counterpart. These molecules termed “combi-molecules” were designed to remain small enough for their quinazoline moiety to be able to bind to the ATP site of EGFR and to block a divergent target such as Src or DNA. Using molecular modeling, a structure-based drug design program was used to identify a linker that could be placed between the quinazoline moiety required for binding in the ATP site and the appendage directed at the divergent target. A solvent exposed ASP residue near the opening of the EGFR ATP binding pocket was used to optimize interaction with ionizable linkers. This interaction was found to be tolerant of bulky substituents and was exploited to append other pharmacophores to the combi-molecules. The results showed that in the category of mixed EGFR-DNA targeting molecules, the EGFR inhibitory potency of EGFR was in the low micromolar to nM range and the compounds also retained significant DNA damaging potential. In the category of EGFR-Src targeting molecules, we identified SB163 that contained a quinazoline moiety, the ionizable spacer and an analogue of PP2. SB163 showed significant antiproliferative and antimetastatic property in a Boyden Chamber assay and its activity was superior to that of a combination of known Src inhibitor PP2 + ZD1839, a clinical inhibitor of EGFR. Furthermore, sub-cellular distribution studies, using a fluorescent probe containing the optimized ionizable spacer showed that their biodistribution is unique with preferential localization in the perinuclear region. The results in toto suggests that in the design of promiscuous molecules, an ionizable basic arm linked to the aminoquinazoline moiety preserves EGFR inhibitory potency and allow freedom to append other moiety directed at the cross- target. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A142.