ALBERT

Description: Targeted T-cell therapy is a potentially less toxic strategy than allogeneic stem cell transplantation for providing a cytotoxic antileukemic response to eliminate leukemic stem cells (LSCs) in acute myeloid leukemia (AML). However, this strategy requires identification of leukemia-associated antigens that are immunogenic and exhibit selective high expression in AML LSCs. Using microarray expression analysis of LSCs, hematopoietic cell subpopulations, and peripheral tissues to screen for candidate antigens, cyclin-A1 was identified as a candidate gene. Cyclin-A1 promotes cell proliferation and survival, has been shown to be leukemogenic in mice, is detected in LSCs of more than 50% of AML patients, and is minimally expressed in normal tissues with exception of testis. Using dendritic cells pulsed with a cyclin-A1 peptide library, we generated T cells against several cyclin-A1 oligopeptides. Two HLA A*0201-restricted epitopes were further characterized, and specific CD8 T-cell clones recognized both peptide-pulsed target cells and the HLA A*0201-positive AML line THP-1, which expresses cyclin-A1. Furthermore, cyclin-A1–specific CD8 T cells lysed primary AML cells. Thus, cyclin-A1 is the first prototypic leukemia-testis-antigen to be expressed in AML LSCs. The pro-oncogenic activity, high expression levels, and multitude of immunogenic epitopes make it a viable target for pursuing T cell–based therapy approaches.

Description: Wilms’ Tumor 1 is a transcription factor found to be recurrently mutated (WT1mut) in 10% of normal karyotype acute myeloid leukemia (NK-AML), predominantly in young adults with intermediate-poor prognosis, often in association with FLT3-ITD and high white cell count. Mutations are usually heterozygous and consist of small insertions or deletions clustered around exons 7 and 9, which encode zinc-finger DNA binding domains. In general, mutations disrupt these DNA binding domains leading to a truncated protein that may act in a dominant negative fashion. Currently, the mechanism by which WT1mut contributes to leukemogenesis is unknown, and no lead drug targets linked to this mutation have been identified to date. Using a novel computational method based on Boolean implications that link the presence of a somatic mutation to CpG methylation on a site-by-site basis, we found that mutation in WT1 is strongly linked to DNA hypermethylation in AML patient samples. In order to validate these findings, we expressed mutant WT1 protein prematurely truncated at exon 7 in THP-1 AML cells (confirmed to be wildtype for WT1 at both alleles), and after 10 passages we measured DNA methylation by 450K bead-chip arrays. We found consistent upregulation of DNA methylation in mutant but not wildtype WT1-expressing cells when compared to parental THP-1 cells, validating WT1 mutation as an active driver of DNA hypermethylation. Additional methylome analysis of human hematopoietic stem and progenitor compartments (HSPC) including (HSC, MPP, L-MPP, CMP, and GMP) indicated that WT1mut induces predominantly de novo DNA methylation, as virtually all CpG sites induced by the mutant protein are unmethylated in normal HSPC. Strikingly, the pattern of methylation in both WT1mut patient samples and WT1mut-THP-1 cells was enriched for polycomb repressor complex 2 (PRC2) target genes (p

Description: IDH1 and IDH2 are two of the most frequently mutated genes in acute myeloid leukemia at an overall frequency of about 15-20%. The genes encode enzymes in the citric acid cycle that normally catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG). The mutant enzymes gain a neomorphic activity that catalyzes the conversion of α-KG to (R)-2-hydroxyglutarate (2-HG). The intracellular concentration of (R)-2-HG is over 100-fold higher in IDH-mutated cells than in wildtype cells. (R)-2-HG has been shown to be a competitive inhibitor of multiple α-KG dependent dioxygenases including TET2, and the Jumonji-C domain containing histone demethylases. These enzymes are thought to be the main targets through which (R)-2-HG exerts its effects on leukemogenesis. We previously reported that inhibition of the anti-apoptotic BCL-2 protein is synthetic lethal against mutant IDH which we discovered through a large-scale pooled lentiviral RNA interference screen. We confirmed that expression of mutant IDH1 or IDH2 strikingly sensitized AML cells to shRNA-mediated BCL-2 knockdown and pharmacologic BCL-2 inhibition with ABT-199, a highly specific BH3 mimetic. Importantly, we found that primary human AML blasts harboring IDH mutations were significantly more sensitive to ABT-199 than blasts with wildtype IDH ex vivo and in xenograft transplant models. Furthermore, we showed that ABT-199 was able to target the leukemic stem cell compartment in IDH-mutated samples. Here, we present our work to uncover the synthetic lethal mechanism. An important clue to the mechanism surfaced with the finding that treatment with a cell-permeable precursor of (R)-2-HG sensitized AML cells to BCL-2 inhibition, indicating that the intracellular accumulation of (R)-2-HG found in IDH-mutated cells is sufficient to mediate the phenotype. In addition, we found that (R)-2-HG was able to sensitize isolated mitochondria to ABT-199 with collapse of the mitochondrial transmembrane potential as a surrogate marker for commitment to apoptosis. This finding indicates that 1) the target mediating the synthetic lethal phenotype is localized to the mitochondria, and 2) changes in the epigenome and expression of nuclear-encoded genes are not required for the synthetic lethal phenotype. To identify the potential mitochondrial molecular target, we focused our analysis on the effect of (R)-2-HG on the enzymatic activity of individual complexes in the electron transport chain (ETC), given that ETC dysfunction can potentially alter the threshold for apoptosis. We found that (R)-2-HG at concentrations found in IDH mutated cells inhibited the in vitro enzymatic activity of complex IV (cytochrome C oxidase (COX)) in a dose-dependent manner, but had no effect on the remaining ETC complexes. This finding has in vivo significance as COX activity in intact IDH-mutated primary AML cells was found to be significantly decreased compared with non-mutated AML cells. Next, we investigated the possibility that COX inhibition is sufficient to induce BCL-2 dependence. We found that suppression of COX activity with chemical inhibitors or genetically through shRNA-mediated knockdown of a COX subunit (COX-IV) was sufficient to sensitize AML cells to ABT-199. Furthermore, treatment with tigecycline, a FDA-approved antibiotic that has previously been shown to disrupt ETC function through inhibition of mitochondrial translation resulting in decreased expression of mitochondrial-encoded proteins including the catalytic subunits of the COX complex, reproduced the sensitization effect in non-IDH mutated AML cells. Based on the above findings, we propose a mechanistic model in which (R)-2-HG accumulation in IDH mutant cells directly inhibits COX, thereby lowering the mitochondrial threshold for triggering apoptosis upon BCL-2 inhibition. In summary, we discovered that in addition to the previously described inhibition of α-KG dependent dioxygenases through (R)-2-HG production, IDH mutations also affect mitochondrial bioenergetics. This finding opens up the intriguing possibility that IDH mutations contribute to leukemogenesis not only through epigenetic changes but also through metabolic dysregulation. Lastly, our findings form the rational basis for combining agents that disrupt ETC function such as tigecycline with ABT-199 to target resistant cancer cells and maximize the clinical utility of this promising drug. Disclosures Medeiros: Agios: Consulting - Ad board Other.

Description: Acute myeloid leukemia (AML) is a hematologic malignancy initiated by leukemia-initiating or leukemia stem cells (LSC) which can differentiate into clonally related leukemic blast cells. This leukemia stem cell model proposes that functional properties of LSC and their blast progeny must be derived by epigenetic differences. Here, we examined genome wide DNA methylation of LSC-enriched populations and blast cells from 15 AML patients, along with 6 well-defined hematopoietic stem and progenitor cell (HSPC) populations from 5 normal controls using Illumina Infinium Human Methylation 450 BeadChip array. Strikingly, LSC-enriched populations exhibited global hypomethylation compared to non-engrafting blast cells, demonstrating that epigenetic change could drive the functional difference of LSC and their blast progeny. We defined an LSC epigenetic signature by integrating DNA methylation and gene expression analysis. The signature independently predicted overall survival of patients in both DNA methylation and gene expression data sets. Finally, we identified that LSC-enriched populations formed two major clusters when compared to normal HSPC: a granulocyte-macrophage progenitor (GMP)-like and a lymphoid-primed multipotential progenitor (L-MPP)-like subgroup that may reflect the cell of origin for these cases. These subgroups showed strong association with cytogenetic abnormalities and molecular mutations associated with the cell of origin. These results provide the first evidence for epigenetic variation between LSC and their blast progeny that are prognostic, and for epigenetically defined cell of origin of AML LSC. Disclosures No relevant conflicts of interest to declare.

Description: Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibody-mediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRPα and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors.

Description: Abstract 638 Relapse is a major cause of treatment failure in adults with Acute Myeloid Leukemia (AML) and new treatments are required. DNA methyltransferase inhibitors (DNMTi) (e.g. 5-Azacitidine (AZA)) alone or in combination with histone deacetylase inhibitors (HDI) (e.g. sodium valproate, (VAL)) represent a potentially important therapeutic advance. However, their clinical utility is limited by the short median response duration and relapse is almost universally observed. It has been postulated that chemoresistant leukemic stem/progenitor cells (LSC) contribute towards disease relapse but this hypothesis has never been examined clinically. We therefore correlated clinical outcomes in 79 patients with AML or myelodysplasia (MDS) ineligible for myelosuppressive chemotherapy who were treated with combined AZA and VAL with serial immunophenotypic and functional analysis of bone marrow stem/progenitor cells(1). Clinical and marrow response was assessed at 1, 3 and 6 months and clinical responses assessed according to Cheson criteria. 64 patients had AML and 57 had received prior intensive chemotherapy. 49 patients completed ≥3 cycles of AZA. Major clinical responses (MCR) (defined as acquisition of CR, CRi or PR) were observed in 26/79 patients (14 CR/Cri, 12 PR). MCR was achieved with a median of 2 cycles of therapy (range 1 to 4). 52% of patients who received ≥3 cycles of AZA achieved an MCR. In patients achieving a CR/CRi median survival was 18.2 months compared to 5 months for patients who either failed to respond or only achieved a PR (p=

Description: Key Points Akt/FLNA/TIF-90 signaling regulates rRNA synthesis in acute myelogenous leukemia cells. Direct targeting of Akt has potential therapeutic applications in acute myelogenous leukemia treatment.

Description: Abstract 4 Acute myeloid leukemia (AML) is an aggressive malignancy of hematopoietic progenitors with poor clinical outcomes. Despite the power of next-generation genome sequencing to describe AML genomes and to identify recurrent mutations, our fundamental understanding of the genomics of leukemogenesis is incomplete. Founding mutations in the majority of AML cases are largely unknown because pre-leukemic cells are clinically silent and are outcompeted by their malignant descendants. Our limited knowledge of founding mutations comes from infrequent cases of AML arising secondary to antecedent clonal bone marrow disorders or rare instances of inherited syndromes, but this does not include the large majority of de novo AML cases. Previously, we showed that non-leukemic hematopoietic stem cells (HSC) contain clonal antecedents of AML in patients in long-term remission post-therapy, and have proposed a model in which serial acquisition of mutations occurs in self-renewing HSC. More recently, we demonstrated the prospective separation of residual HSC from AML cells, based on differential expression of surface markers such as CD47 and TIM3.1,2 Here, we investigated this model and the nature of founder mutations through the genomic analysis of de novo AML and patient-matched residual non-leukemic HSC, speculating that these residual non-leukemic HSC might in fact constitute a reservoir of pre-leukemic HSC harboring founder mutations, but lacking the complete complement of abnormalities required to generate AML. Using exome sequencing, we identified mutations present in multiple individual AML genomes (mean 10 mutations per patient) and screened for them in the residual HSC. In most cases, we identified several mutations present in residual HSC that retained normal multilineage differentiation in vivo. These “early” mutations include NPM1c and novel AML mutations in genes involved in the cell cycle and mRNA biogenesis. Putative “late” mutations absent from residual HSC and only found in leukemic cells include FLT3 ITD and IDH1 R132H. Next, using custom-designed SNP Taqman genotyping assays, we analyzed single residual HSC for the presence of the identified “early” mutations. As hypothesized, we determined that a clonal progression of mutations occurs in non-leukemic HSC, based on the identification of individual cells containing subsets of these “early” mutations. Quantitative genetic analyses of the HSC compartment enabled us to reconstruct the subclonal architecture of normal and pre-leukemic stem cells. In all cases, normal HSC were 6–50 times more numerous than pre-leukemic HSC, and in one case where we identified two sequential populations of pre-leukemic HSC, the less mutated population was 25 times more numerous than its more mutated descendent. This result contrasts with the classical model of a linear succession of increasingly dominant pre-leukemic subclones, suggesting that the relationship between subclone size and clonal progression may be complex. In summary, our results show that pre-leukemic HSC reveal the clonal evolution of AML genomes from founder mutations. Ultimately, these clonal antecedents of leukemia may prove to be clinically important. Indeed, some cases of relapsed pediatric ALL have been shown to arise from a clone ancestral to the presenting leukemia. The same may be true in AML, in which relapsed disease develops from a pre-leukemic HSC clone that acquires additional novel mutations resulting in a genetically divergent leukemic relapse. This possibility suggests that pre-leukemic HSC constitute a cellular reservoir that may need to be targeted for more durable remissions. Disclosures: No relevant conflicts of interest to declare.

Description: The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant—driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114neg/lo compartment, and almost completely absent in the CD114pos subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.