Single-Cell Transcriptomic and Proteomic Analysis of Acute Myeloid Leukemia (AML) Patients with Abnormalities on Chromosome 7

Abstract Background: Chromosome 7 (chr7) abnormalities are commonly seen in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome and are associated with poor prognosis. Flow cytometry (FCM) is typically used clinically to quantify residual malignancy during treatment but the relationship of cell surface immunophenotype with genetic features is incompletely defined. Single-cell RNA sequencing (scRNA-seq) with oligonucleotide-conjugated antibodies may be able to integrate cytogenetic genotypes found within leukemic clones with specific transcriptomic and immunophenotypic signatures. Methods: Bone marrow (BM) aspirate was collected from 12 AML patients with known abnormalities on chr7 according to cytogenetics and 3 healthy donors (HDs). We previously established a reference for normal immune cells where we assessed the BM of 20 HDs (PMID:30518681). HDs in present study were selected from this same cohort. Multi-parameter FCM was performed as previously described and scRNA-seq with 31 antibodies (10x Genomics, 3'v2) was performed on BM mononuclear cells. Data from 16 samples (including 1 patient replicate) were mapped onto the atlas with dimension reduction in gene expression (GEX) by principal component analysis and Uniform Manifold Approximation and Projection (UMAP). Cell clusters were constructed and surface proteins were utilized to determine cellular annotation. Patient GEX profiles were compared to those of HDs, and two algorithms were used to identify malignant cells with chromosomal abnormalities. Alterations of large chromosomal segments were identified by Hidden Markov Model. Clinical cytogenetics and expression matrices from the model output were combined to annotate individual cells as malignant or normal for all potential regions. Machine learning classifiers were applied to predict malignant cells using protein expression and important proteins were selected by models with measure for significant features. Results: After quality control, 132,658 cells were included, of which 43,441 and 12,572 cells from AML patients and HD respectively had additional cell surface immunophenotyping data. Samples were well-integrated by UMAP (Figure 1A) and cell annotation overlapped with previously reported annotations by GEX of HDs (Figure 1B), with 15,270 malignant cells identified (Figure 1C). Malignant groups were created if more than 20 cells shared the same chromosomal alterations. Among all 12 patients, 3 of them only had monosomy 7 as the sole genetic aberration, while 9 patients had abnormalities on other chromosomes. The correlation between cell types showed distinct features among patients, and cells in different groups had different protein expression profiles. For example, 22% of the cells (n = 432) from the myeloid population of patient 1 (n = 1957) were identified as normal and 77% had a loss of chr7 (Figure 2A). Clustering in lower resolution separated them into two groups: CD11b+CD33+ (n = 1026) and CD133+CD34+ (n = 931). The CD133+CD34+ group had a higher percentage of malignant cells overall (74% vs. 83%) while a subset of the CD11b+CD33+ group (CD16+CD13+) had the highest percentage of cells with losses on both chr2 and chr7 (60%). Data from patient 2 also suggested that cells with different immunophenotypes were estimated to have different chromosomal changes (Figure 2B). While 69% of the malignant cells had a loss on chr7, most of those without changes on chr7 were from CD45+CD11b+ group where 62% of the cells did not have a detectable change on chr7. The top significant proteins for distinguishing malignant from normal cells among all patients were CD117, CD33, CD34, CD44, and CD47. FCM intensity was compared with the scRNA-seq immunophenotyping data, confirming the similarity in distribution. The scRNA-seq data with immunophenotyping can capture and recapitulate the leukemic immunophenotype, further linking it with copy number changes to reveal potential subclonal structures. Conclusion: By comparing the expression profile of AML patients with abnormalities on chr7 to HDs, this study provides evidence that leukemic immunophenotype is correlated with chromosomal structural changes. The experiments on a single-cell level were able to identify clones in higher resolution and revealed potential cell surface protein markers that could be used clinically to identify specific malignant populations in patients with myeloid malignancies. Figure 1 Figure 1. Disclosures Ghiaur: Syros Pharmaceuticals: Consultancy; Menarini Richerche: Research Funding. Hourigan: Sellas: Research Funding.

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Highly Multiplexed Cell Surface Immunophenotyping with Genotyping and Concurrent Transcriptomic Analysis of NPM1 mutated Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2021-151366 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1288-1288

Author(s):

Devdeep Mukherjee ◽

Gege Gui ◽

Laura W. Dillon ◽

Christopher S. Hourigan

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Cell Surface ◽

Single Cell ◽

Myeloid Leukemia ◽

Type A ◽

Patient Specific ◽

Malignant Cells ◽

Surface Proteome ◽

Acute Myeloid

Abstract BACKGROUND: The pathogenesis of acute myeloid leukemia (AML) is often attributed to the presence of somatic allelic variant(s) in hematopoietic stem/progenitor cells. However, malignant clones may have heterogenous cell-surface immunophenotypes including overlap with non-malignant cells. While leukemia-associated immunophenotypes and difference from normal approaches are used for flow cytometric assessment during and after treatment, such analysis may underrepresent true leukemia disease burden. Assessments of AML measurable residual disease (MRD) using flow cytometry and molecular methods have been reported as discrepant. Single-cell RNA sequencing experiments have recently attempted to distinguish malignant cells based on gene expression and/or immunophenotypic profiles alone. We hypothesized that single-cell genotyping of mutated transcript(s) coupled with broad surface proteome and transcriptome profiling could provide an integrated multimodal method for AML characterization. METHODS: We adapted the previously reported "genotyping of transcriptomes" (PMID: 31270458) to identify cells carrying the NPM1 type A mutation commonly seen, and typically stable throughout the disease course, in AML. Healthy human peripheral blood mononuclear cells (PBMC) were mixed with an AML cell line carrying NPM1 type A mutation (OCI-AML3) at 7:3 ratio and labelled with 163 oligo-tagged antibodies. Single cell 3'v3 gene expression- (GEX), antibody derived tag- (ADT) and genotyping of NPM1 (GNPM) -libraries (10X Genomics) were sequenced on the NovaSeq 6000 (Illumina). Results were processed using Seurat 4.0 toolkit. RESULTS: In total, 72% (n=1680) of barcoded cells could be genotyped for NPM1. Of the genotyped cells, 59% (n = 986) were not NPM1 mutated. Visualization using Uniform Manifold Approximation and Projection (UMAP) showed separation of healthy PBMCs and OCI-AML3 cells using protein data, confirmed by annotation using NPM1 genotyping (Figure 1). We found a significant positive correlation between mRNA and corresponding cell surface protein expressions in non-mutated (Pearson's coefficient, r = 0.502, p = 6.87e-11) and NPM1 mutated (r= 0.392, p= 7.5e-7) cells. Compared to non-mutated, NPM1 mutated cells showed nearly 14-fold higher NPM1 transcript levels. In addition, a total 63 proteins were highly expressed on the surface of NPM1 mutated cells (Figure 2). Among these, CD33 and CD36 showed maximum 8-fold increase in expression. Other highly expressed proteins with at least >2.5-fold change were cell adhesion molecules (including CD328, CD155, and CD56), extracellular matrix binding proteins (CD49a/b) and interleukin receptor (CD123). CONCLUSION: Overall, our results demonstrate proof of principle that high-throughput cell surface proteome, transcriptome and genotyping analysis can be simultaneously performed to comprehensively and confidently characterize individual AML cells. Patient-specific multiomics data with broad cell-surface proteomic screening may allow novel target identification for monitoring and/or therapeutic intervention. Ongoing work will now use this methodology to characterize a cohort of NPM1 mutated AML patient samples. Figure 1 Figure 1. Disclosures Hourigan: Sellas: Research Funding.

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Single-Cell Mass Cytometry Characterizes Phenotypic and Functional Heterogeneity in Acute Myeloid Leukemia at Diagnosis, in Remission and Relapse

Blood ◽

10.1182/blood-2018-99-111970 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 912-912

Author(s):

Lina Han ◽

Peng Qiu ◽

Jeffrey L. Jorgensen ◽

Duncan Mak ◽

Jared K Burks ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Intracellular Signaling ◽

Myeloid Leukemia ◽

Research Funding ◽

Cell Mass ◽

Mass Cytometry ◽

Intracellular Signaling Pathways ◽

Genetics Research ◽

Acute Myeloid

Abstract Patients with acute myeloid leukemia (AML) continue facing poor long-term survival due to high relapse rate. Persistence of dormant self-renewing leukemia stem/progenitor cells (LSPCs) has been implicated as driver of subsequent relapse, and stem cell gene signatures are associated with poor outcome (Shlush et al. Nature 2017; Ng et al. Nature 2016; Eppert et al Nat Med 2011). Identification of the unique phenotypes and functional proteins in LSCs surviving induction therapy may aid in understanding the mechanisms of chemoresistance and provide novel therapeutic targets in the residual leukemia clones. In this study, we have developed and optimized a comprehensive single-cell mass cytometry (CyTOF) panel, including 36 markers to define LSPCs, with the goal to identify and characterize expression of multiple intracellular signaling pathways and anti-apoptotic proteins in residual AML cells. The validated CyTOF panel was applied in 21 samples collected from 7 AML patients at diagnosis, in remission and at relapse and 5 healthy donors. Data were analyzed using SPADE (Qiu et al, Nat Biotechnol 2011) or Cytofkit (Chen et al. PLoS Comput Biol 2016) tools. We first generated SPADE trees for all diagnostic samples (n=7, Fig 1A), annotating 7 distinct cell populations based on the median expression of selected surface markers as shown in the heatmap (Fig 1B). From these annotations, populations A1, A2 and A6 were positive for CD34 expression, with A6 representing phenotypically the most primitive fraction CD34hiCD38low population (frequency range 0.04%-17.32%). Fractions A1 and A2 expressed more committed myeloid markers, positive for CD135 and CD33 progenitor markers and differentiation markers including CD15, CD11b, and CD7 (Fig 1B). Variability was observed in terms of cell composition, and non-stem fractions A3-A5 were abundant populations in all AML samples except AML2. We further studied the activation/expression of functional proteins in these populations and found that pro-survival BCL-2 protein was highly expressed in the primitive A6 population across AML samples (median intensity 9.0 ± 5.3 in A6 vs 3.5 ± 3.9 in other populations). Variable p-AKT activation was observed in both A6 (4.9 ± 4.2) and differentiated A3-A5 populations (4.5 ± 3.6). We next examined how multi-parametric CyTOF analysis will aid in characterization of MRD populations by comparing samples from 7 patients collected at the time of diagnosis, in remission and at relapse. Using the Cytofkit bioconductor analysis and FlowSOM algorithm, we identified distinct patterns of relapse (Fig 1C). In AML 1-3, major populations were markedly reduced by induction chemotherapy, but residual cells re-grew and contributed to relapse. In AML 4-7, the major populations at diagnosis were eliminated by the therapy, but minor (or undetectable) populations at diagnosis progressed over treatment and represented the bulk of leukemia upon relapse. This finding is consistent with genomic studies that relapse may originate from either the founding clones or subclones that acquire additional mutations (Ding et al. Nature 2012). In AML#3, a major population (cluster 7, CD34−CD38+CD123+CD64+HLA-DR+CD99+, 75.6%) present at diagnosis was identified as persisting in remission at 2.5% by CyTOF analysis but not by conventional MRD flow cytometry and gave rise to the overt leukemia (78.1%) at relapse (Fig 1D). In this patient whose AML harbored mutation in negative MAPK regulator phosphatase PTPN11, persistent AML cells expressed BCL-2, MCL-1, and p-p38MAPK (Fig 1E), consistent with dominant activation of MAPK signaling and anti-apoptotic proteins. We found highly enriched BCL-2 expression and p38MAPK activation in relapse-driving clones in AML5 and AML7, and in diagnostic clone in AML6 (Fig 1F). In summary, using CyTOF, SPADE and Cytofkit analysis tools, we characterized LSPC-specific intracellular signaling pathways in AML samples at diagnosis, in remission and at the time of relapse. Distinct populations were identified to contribute to relapse, indicating that use of additional targeted therapies such as BCL-2 inhibitors may be instrumental post remission to prevent relapse. In conclusion, analysis of the multi-parametric single cell CyTOF mass cytometry may aid in understanding clonal evolution during chemotherapy and identify potential therapeutic targets in individual patients. Disclosures Ravandi: Astellas Pharmaceuticals: Consultancy, Honoraria; Xencor: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Abbvie: Research Funding; Jazz: Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Macrogenix: Honoraria, Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Orsenix: Honoraria; Jazz: Honoraria; Orsenix: Honoraria; Sunesis: Honoraria; Sunesis: Honoraria; Seattle Genetics: Research Funding; Macrogenix: Honoraria, Research Funding. Roboz:Roche/Genentech: Consultancy; Pfizer: Consultancy; Bayer: Consultancy; Celgene Corporation: Consultancy; Astex Pharmaceuticals: Consultancy; Argenx: Consultancy; Roche/Genentech: Consultancy; Janssen Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy; Pfizer: Consultancy; Eisai: Consultancy; Jazz Pharmaceuticals: Consultancy; Orsenix: Consultancy; Astex Pharmaceuticals: Consultancy; Aphivena Therapeutics: Consultancy; Celgene Corporation: Consultancy; Sandoz: Consultancy; Novartis: Consultancy; Bayer: Consultancy; Otsuka: Consultancy; Aphivena Therapeutics: Consultancy; Celltrion: Consultancy; Daiichi Sankyo: Consultancy; Sandoz: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Celltrion: Consultancy; AbbVie: Consultancy; AbbVie: Consultancy; Orsenix: Consultancy; Cellectis: Research Funding; Novartis: Consultancy; Cellectis: Research Funding; Argenx: Consultancy; Eisai: Consultancy; Otsuka: Consultancy. Andreeff:AstraZeneca: Research Funding. Guzman:Cellectis: Research Funding. Konopleva:Stemline Therapeutics: Research Funding.

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Clonal Heterogeneity in Differentiation Response and Resistance to the IDH2 Inhibitor Enasidenib in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.724.724 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 724-724

Author(s):

Lynn Quek ◽

Muriel David ◽

Alison Kennedy ◽

Marlen Metzner ◽

Michael Amatangelo ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Myeloid Leukemia ◽

Research Funding ◽

Functional Heterogeneity ◽

Employment Equity ◽

Sequential Samples ◽

Equity Ownership ◽

Acute Myeloid ◽

Refractory Aml

Abstract Background Mutations in Isocitrate Dehydrogenase 2 (IDH2) occur in many cancers including Acute Myeloid Leukemia (AML). In preclinical models mutant IDH2 (mIDH2) causes partial hemopoietic differentiation block1. Recently, we showed that single agent enasidenib, a first-in-class, selective mIDH2 inhibitor, produces a 40% response in relapsed/refractory AML patients by promoting differentiation2. Here, we studied response and acquired resistance to enasidenib, in sequential samples treated in the Phase 1 study of Enasidenib in relapsed/ refractory AML patients. Results We studied a cytogenetically and genetically representative subset of 25 patients enriched for enasidenib responders, genotyped by whole exome sequencing (WES) or cancer gene panel targeted re-sequencing. Pre-enasidenib, differentiation arrest in these AML patients resulted in abnormally expanded leukaemic progenitors or precursors and diminished mature haematopoietic populations. Complete remission (CR) post-enasidenib was associated with in increased mature populations, near-normalisation of haematopoietic progenitor profiles, and restoration of in vitro progenitor function. In most patients, mature blood cells (of erythroid and granulocyte-monocyte lineages) post-enasidenib are IDH2 mutant, consistent with enasidenib inducing differentiation of IDH2 mutant leukaemic progenitors/ precursors. Each mIDH2 patient studied had on average 13 somatic, non-synonymous exonic or splice site mutations in addition to IDH2 . We used single cell genotyping (SCG) to reveal linear or branching clonal structures in mIDH2 AML. We combined clonal structure data and immunophenotyping of haematopoietic progenitor, precursor and mature populations to track functional behaviour of mIDH2 clones before, and during enasidenib treatment. We demonstrate, for the first time, that mIDH2 subclones within the same patient are functionally heterogeneous: both in their ability to differentiate pre-enasidenib, and in their sensitivity to Enasidenib-induced differentiation. This suggests that different combinations of co-operating mutations result in functional heterogeneity of mIDH2 clones. When we studied the contribution of mIDH2 clones to functional haematopoiesis at CR, we found that this was supported by either ancestral or leukaemic terminal mIDH2 clones. Despite a median survival of 18-21 months in patients who respond to enasidenib, most patients eventually relapse3. In contrast to targeted therapies such as tyrosine kinase inhibitors, in all 12 relapse samples studied, none had second site mutations in IDH2 . Furthermore, 2-hydroxyglurate (2HG) levels remain suppressed in most patients suggesting enasidenib remains effective in inhibiting mIDH2 enzyme. Instead, mIDH2 clones, which had persisted at CR or partial remission (PR) acquired additional mutations or aneuploidy, highlighting bypass pathways which re-impose differentiation arrest. We found 4 patterns: i) acquisition of IDH1 codon R132 mutations which resulted in a rise in 2HG (n=2), ii) deletion of chromosome 7q (n=4), iii) gain of function mutations in genes implicated in cell proliferation (FLT3, CSF3R) (n=3) and iv) mutation in hematopoietic transcription factors (GATA2, RUNX1) (n=2). We also found mutations in 4 genes (DHX15 and DEAF1 (n=1) ; NFKB1 (n=1) and MTUS1 (n=1)) not previously implicated in haematopoietic differentiation arrest which were selected for, or evolved in mIDH2 subclones at relapse. Conclusion This study provides a paradigm of how deep clonal single cell analysis in purified hemopoietic compartments in sequential samples through therapy reveals clonal complexity and the impact of the selective pressure of therapy on clonal architecture. Furthermore, we gain insights into the functional heterogeneity of mIDH2 subclones in their ability to differentiate pre-and post-Enasidenib. Further analysis of this kind in a larger cohort of IDH2 -inhibitor-treated patients would also provide insight to improve efficacy of this novel class of therapeutics, and design of combination therapies in AML and other cancers. Finally, this provides a platform for further study of the pathways mediating enasidenib resistance. References 1. Kats, L.M. , et al. Cell Stem Cell14, 329-341 (2014). 2. Amatangelo, M.D. , et al. Blood (2017). 3. Stein, E.M. , et al. Blood (2017). Disclosures Quek: Celgene Corporation: Research Funding. Amatangelo: Celgene Corporation: Employment. Agresta: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yen: Agios: Employment, Equity Ownership. Stein: Pfizer: Consultancy, Other: Travel expenses; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Constellation Pharma: Research Funding; Novartis: Consultancy, Research Funding; GSK: Other: Advisory Board, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding. De Botton: Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Levine: Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding. Vyas: Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

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Efficacy and Effectiveness of Azacitidine In Elderly Patients with Acute Myeloid Leukemia and Blasts Counts below 30%: Experience of the Spanish Registry

Blood ◽

10.1182/blood.v116.21.4354.4354 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4354-4354

Author(s):

Regina García ◽

Monserrat Arnar ◽

Elisa Luño ◽

Joan Bargay ◽

Ma Mar Romero ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Elderly Patients ◽

Myeloid Leukemia ◽

Research Funding ◽

De Novo ◽

Previous Treatment ◽

Similar Rate ◽

Monosomy 7 ◽

Duration Of Response ◽

Acute Myeloid

Abstract Abstract 4354 Introduction: The incidence of acute myeloid leukemia (AML) increases exponentially with age. Elderly patients normally have a previous hematologic condition like myelodysplastic syndrom (MDS), cytogenetic abnormalities that correlate with resistance to conventional chemotherapy schemes and a shorter duration of response. Not only responses are worse, also the hospitalization duration is longer, and the mortality associated to the treatment is higher (30%). Therefore, treating these patients with standard chemotherapy schemes is associated, in many cases, to a low probability of obtaining satisfactory results. Introducing less toxic and/or more efficient agents can change the course of the illness for this sub-group of patients. Azacitidine (AZA) is a hypomethylating drug recently approved in Europe for the treatment of AML with 20 – 30% blasts. Materials and methods: We present clinical data analysis of a longitudinal, multicenter and retrospective Spanish patients’ registry of patients with de novo and relapse AML. Data on the course of the disease and managment of patients with de novo and relapse AML with low blasts count (20-30%) treated with AZA under compasionate use were analized. Result: Twentyfive patients (male/female 13/12) with a median age of 67 years with AML were included in the registry. Ten patients (40%) had refractory/relapsed AML and had received previous treatment with intensive chemotherapy. Cytogenetic alterations were reported in 10 patients (4 monosomy-7). An average of 7 cycles of AZA were administered to the patients (range 1–25). The most frequently used dose was 75 mg (82%), of which 28% received a scheme of 5 days, 24% received a scheme of 5+2 and 48% of 7 days. The global responses were of 56% with 20% of complete responses. The most frequent toxicity described was hematologic. Conclusion: Our data corroborates, with a similar rate of responses, what Journal of Clinial Oncology published about the sub analysis of the AZA-001. 5-Azacitidine is an efficient and safe drug in patients with AML. Good response rates and a toxicity and mortality related to the acceptable treatment in this sub-group of patients. Disclosures: García: Celgene: Research Funding. Bargay:Celgene: Research Funding.

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Decitabine Response Associated Gene Expression Patterns In Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.3756.3756 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3756-3756 ◽

Cited By ~ 2

Author(s):

Stephan R Bohl ◽

Rainer Claus ◽

Anna Dolnik ◽

Richard F. Schlenk ◽

Konstanze Döhner ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Acute Myeloid Leukemia ◽

Immune System ◽

Cell Surface ◽

Myeloid Leukemia ◽

Research Funding ◽

Expression Patterns ◽

Gene Expression Patterns ◽

Acute Myeloid

Abstract The hypomethylating agent decitabine (DAC) represents a therapeutic option for acute myeloid leukemia (AML) patients who are not eligible for an intensive treatment regime. However, there are no biomarkers available yet that can predict patients who will likely benefit from this epigenetic therapy. Therefore, we executed a gene expression analysis prior to the treatment of patients with DAC in order to evaluate gene expression patterns associated with response to DAC that ultimately might be used to predict DAC outcome. Patients had been entered in a multicenter phase II trial of DAC as first-line treatment of older AML patients judged unfit for induction chemotherapy (Lübbert et al. Haematologica 2011; NCT00866073). Gene expression was profiled in selected DAC responders (n=17) and non-responders (n=19; non-response was defined as stable disease or progressive disease). These groups did not show significant differences regarding age, gender, performance status, blast counts and cytogenetics. Supervised data analysis strategies were applied to identify genes and gene patterns associated with DAC response. While the study cohort comprised a heterogeneous group of AML patients, a class comparison analysis nevertheless could reveal a DAC response associated gene pattern comprising 301 genes at a significance level of p<0.05. This signature was enriched for genes belonging to pathways that are essential in immune response and tumor suppressor function. Among these genes that were significantly associated with no DAC response included IFI44L, IFI27, PDK4, MX1, FAS, and ITGB2; in contrast to SLC24A3, MUM1, TNFSF9, DBN1, ABAT, and DDX52, which were significantly higher expressed in patients that showed response to DAC treatment. Significantly over-expressed in the DAC non-responder group, the immune and inflammation-related genes IFI44L and IFI27 might reflect a hyperstimulated, but insufficient immune system as has been recently shown in myelofibrosis. As DAC was shown to have the capability to induce cancer testis antigens, thereby generating an efficient immune response with tumor cell lysis by CD8+ T-lymphocytes, an impaired immune system may prevent response to DAC. Furthermore, the non-response signature contained known poor prognostic markers such as PDK4, which has been associated with EVI1 and FLT3-ITD mediated signaling. In addition, we observed high expression of MX1 and FAS in the non-response group. Notably, both genes have been shown to be repressed by promoter hypermethylation in distinct AML subtypes and DAC treatment was able to upregulate their expression levels. In contrast, high pre-treatment expression levels might indicate that in the respective AML cases deregulated promotor methylation might not be the prominent pathomechanism, and thus these cases might less likely benefit from DAC treatment. Finally, we found ITGB2, encoding for an integral cell-surface protein participating in cell-surface mediated signaling, associated with DAC resistance. As recently ITGB3, another member of this integrin protein family, was shown mandatory for leukemogenesis, but not relevant for normal hematopoiesis, high expression of ITGB2 might also play a role in AML and point to leukemias where epigenetic deregulation at the DNA level seems to be a less prone pathomechanism. Among the group of genes linked with response to DAC treatment TNFSF9 can act as cytotoxic leukemic specific T-cell inducer, which has previously been correlated with unfavorable AML subtypes and poor outcome. However, due to the immunomodulation of DAC it seems that the poor prognostic impact of TNFSF9 might be overcome by DAC, thereby rendering TNFSF9 a positive marker for DAC response. In accordance, we found that several genes of the 4-1BB-dependent immune response pathway, including TNFSF9, were more highly expressed in DAC responding patients. Finally, MUM1 encodes also a gene important for interferon dependent immune response, thereby further underscoring a potential immunomodulation effect of DAC. In summary, we were able to elucidate a gene signature which could be used to predict response to DAC treatment in AML. While this gene expression pattern included many genes involved in the immune response, thereby suggesting that the DAC treatment effect is at least in part depending on immunomodulatory effects, further studies are warranted to evaluate the respective markers in larger AML cohorts. Disclosures: Schlenk: Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Chugai: Research Funding; Ambit: Honoraria.

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Single-cell Characterization of Acute Myeloid Leukemia and its Microenvironment Following PD-1 Blockade Based Therapy

10.1101/2020.09.03.278309 ◽

2020 ◽

Author(s):

Hussein A. Abbas ◽

Dapeng Hao ◽

Katarzyna Tomczak ◽

Praveen Barrodia ◽

Jin Seon Im ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Single Cell ◽

Myeloid Leukemia ◽

Cell Subset ◽

Cd8 T Cell ◽

Response To Therapy ◽

Chromosome 7 ◽

Acute Myeloid

AbstractAcute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naïve and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities.SignificanceDetermining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.

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Single-cell map of diverse immune phenotypes in the acute myeloid leukemia microenvironment

Biomarker Research ◽

10.1186/s40364-021-00265-0 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Rongqun Guo ◽

Mengdie Lü ◽

Fujiao Cao ◽

Guanghua Wu ◽

Fengcai Gao ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Myeloid Leukemia ◽

Immune Cell ◽

Immune Surveillance ◽

Cell Types ◽

Developmental Trajectory ◽

Significant Difference ◽

Cell Phenotypes ◽

Acute Myeloid

Abstract Background Knowledge of immune cell phenotypes, function, and developmental trajectory in acute myeloid leukemia (AML) microenvironment is essential for understanding mechanisms of evading immune surveillance and immunotherapy response of targeting special microenvironment components. Methods Using a single-cell RNA sequencing (scRNA-seq) dataset, we analyzed the immune cell phenotypes, function, and developmental trajectory of bone marrow (BM) samples from 16 AML patients and 4 healthy donors, but not AML blasts. Results We observed a significant difference between normal and AML BM immune cells. Here, we defined the diversity of dendritic cells (DC) and macrophages in different AML patients. We also identified several unique immune cell types including T helper cell 17 (TH17)-like intermediate population, cytotoxic CD4+ T subset, T cell: erythrocyte complexes, activated regulatory T cells (Treg), and CD8+ memory-like subset. Emerging AML cells remodels the BM immune microenvironment powerfully, leads to immunosuppression by accumulating exhausted/dysfunctional immune effectors, expending immune-activated types, and promoting the formation of suppressive subsets. Conclusion Our results provide a comprehensive AML BM immune cell census, which can help to select pinpoint targeted drug and predict efficacy of immunotherapy.

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Minimally differentiated acute myeloid leukemia with ring/marker derived from chromosome 7 in a child with Down syndrome

Hematology Transfusion and Cell Therapy ◽

10.1016/j.htct.2018.05.006 ◽

2019 ◽

Vol 41 (1) ◽

pp. 84-87

Author(s):

Maria Luiza Rocha da Rosa Borges ◽

Eliane Maria Soares-Ventura ◽

Thomas Liehr ◽

Terezinha de Jesus Marques-Salles

Keyword(s):

Acute Myeloid Leukemia ◽

Down Syndrome ◽

Myeloid Leukemia ◽

Chromosome 7 ◽

Acute Myeloid

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Discovery of a Novel Dihydroorotate Dehydrogenase Inhibitor That Induces Differentiation and Overcomes Ara-C Resistance of Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood-2019-121869 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2663-2663

Author(s):

Satoshi Kitazawa ◽

Yukiko Ishii ◽

Keiko Makita-Suzuki ◽

Koichi Saito ◽

Kensuke Takayanagi ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Oral Administration ◽

Myeloid Leukemia ◽

Research Funding ◽

Dihydroorotate Dehydrogenase ◽

Once Daily ◽

Hl60 Cells ◽

Pyrimidine Nucleotide ◽

Acute Myeloid

Cancer initiating cells (CIC) are suggested to be responsible for drug resistance and cancer relapse that are associated with poor prognosis. Therefore, drugs effective for CIC could fulfill an unmet clinical need. We performed a drug screen with chemical libraries to find out new compounds which specifically eradicated CIC established in the previous report (Yamashita et al., Cancer Research, 2015). We obtained compounds with a carboxylic acid skeleton as hit compounds. Interestingly, FF1215T, one of the hit compounds, was shown to inhibit growths of CIC by decreasing intracellular pyrimidine nucleotide levels. Finally, we identified dihydroorotate dehydrogenase (DHODH), which was essential for de novo pyrimidine synthesis as the target of the hit compounds in a ligand fishing assay. FF1215T inhibited DHODH enzymatic activity with the 50% inhibitory concentration value of 9 nM, which showed greater potency than well-known DHODH inhibitors brequinar (12 nM), teriflunomide (262 nM), and vidofludimus (141 nM). Growing evidence suggests that DHODH is considered to be a promising target to overcome a differentiation blockade of acute myeloid leukemia (AML) cells (Sykes et al., Cell, 2016).Therefore, we explored the effect of FF1215T on AML growth and differentiation. FF1215T demonstrated growth inhibitory effect in multiple human AML cell lines such as U937, MOLM13, HL60, and MV4-11 with the 50% growth inhibition values of 90-170 nM. FF1215T decreased intracellular pyrimidine nucleotide levels, induced DNA damage marker γ-H2AX possibly due to the replication stress, and finally led to apoptosis in HL60 cells. Cell cycle analysis revealed that FF1215T treatment arrested HL60 and THP1 cells at S phase and increased sub-G1 population in these cells. In addition, our DHODH inhibitors induced upregulation of cell-surface CD11b and CD86, which are monocyte and macrophage differentiation markers, morphological changes, and phagocytic activities in several AML cells, indicating differentiation of AML cells toward monocyte and macrophage by DHODH inhibition. FF1215T also depleted UDP-GlcNAc, a substrate for Protein O-GlcNAcylation, and diminished global O-GlcNAcylation and O-GlcNAcylated protein expressions such as c-Myc, SOX2, and OCT4, which play important roles in maintenance and self renewal of stem cells. We also found that our DHODH inhibitors induced CD11b and CD86, and increased the ratio of macrophage-like cells in primary patient-derived AML cells and these effects were rescued by uridine supplementation (Fig). Inhibitions of colony formations of primary AML cells were also shown after 14 days of FF1215T treatment. In exploring the value of DHODH inhibitors in the clinic, we identified that our DHODH inhibitors worked to overcome the resistance of standard therapy Ara-C. Our DHODH inhibitors were effective against Ara-C-resistant models of HL60 cells as well as HL60 parental cells. Notably, our DHODH inhibitors synergistically inhibited growths of Ara-C-resistant THP1 cells and enhanced CD11b upregulation of THP1 cells when combined with Ara-C by activating conversion of Ara-C to its active form Ara-CTP. Next, we optimized the hit compounds and identified an orally available DHODH inhibitor FF14984T that achieved high and prolonged plasma concentrations in vivo. Oral administration of 10 and 30 mg/kg FF14984T once daily for 10 days exhibited significant anti-tumor effects in mice xenografted with HL60 cells. These treatments showed strong reduction of CTP in tumor and induction of DHO in tumor and plasma. When 30 mg/kg FF14984T was orally administrated to orthotropic MOLM13-xenografted mice once daily for 12 days, hCD45+ cells proportions in bone marrow were decreased whereas hCD11bhigh/hCD45+ ratio increased, indicating that FF14984T induced AML differentiation in vivo. Finally, oral administration of 30 mg/kg FF14984T once daily significantly prolonged survival of mice in U937 orthotropic models. Taken together, we developed a novel potent DHODH inhibitor FF14984T that induced cellular differentiation and anti-leukemic effects on cell lines and primary AML cells. FF14984T is possibly a promising therapeutic option for Ara-C-resistant AML patients that can also benefit from the combination therapy of FF14984T and Ara-C. Identifying the precise mechanism of AML differentiation by DHODH inhibitor and its effects on CIC are currently ongoing. Disclosures Kitazawa: FUJIFILM Corporation: Employment. Ishii:FUJIFILM Corporation: Employment. Makita-Suzuki:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Takayanagi:FUJIFILM Corporation: Employment. Sugihara:FUJIFILM Corporation: Employment. Matsuda:FUJIFILM Corporation: Employment. Yamakawa:FUJIFILM Corporation: Employment. Tsutsui:FUJIFILM Corporation: Employment. Tanaka:FUJIFILM Corporation: Employment. Hatta:FUJIFILM Corporation: Research Funding. Natsume:FUJIFILM Corporation: Research Funding. Kondo:FUJIFILM Corporation: Research Funding. Hagiwara:FUJIFILM Coporation: Employment. Kiyoi:FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Perseus Proteomics Inc.: Research Funding.

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PKR-like Endoplasmic Reticulum Kinase Mediates Apoptosis Induced By Pharmacological AMP-Activated Protein Kinase Activation in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-123364 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2552-2552

Author(s):

Laury Poulain ◽

Adrien Grenier ◽

Johanna Mondesir ◽

Arnaud Jacquel ◽

Claudie Bosc ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Endoplasmic Reticulum Stress Response ◽

Ampk Activation ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Amp Activated Protein Kinase ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a myeloid progenitor-derived neoplasm of poor prognosis, particularly among the elderly, in whom age and comorbidities preclude the use of intensive therapies. Novel therapeutic approaches for AML are therefore critically needed. Adenosine monophosphate (AMP) activated protein kinase (AMPK) is a pleiotropic serine/threonine kinase promoting catabolism that represses anabolism and enhances autophagy in response to stress1. AMPK heterotrimers comprise catalytic α- and regulatory β- and γ-subunits, the latter harboring binding sites for AMP. Targets of AMPK include a host of metabolic pathway enzymes mediating carbohydrate, lipid and protein synthesis and metabolism. Accumulating evidence implicates AMPK in cancer biology, primarily as a tumor suppressor, although minimal AMPK activity may also be required for cancer cell growth under stress conditions2,3. Pharmacological activation of AMPK thus represents an attractive new strategy for targeting AML. We previously used the selective small molecule AMPK activator GSK621 to show that AMPK activation induces cytotoxicity in AML but not in normal hematopoietic cells, contingent on concomitant activation of the mammalian target of rapamycin complex 1 (mTORC1)4. However, the precise mechanisms of AMPK-induced AML cytotoxicity have remained unclear. We integrated gene expression profiling and bioinformatics proteomic analysis to identify the serine/threonine kinase PERK - one of the key effectors of the endoplasmic reticulum stress response - as a potential novel target of AMPK. We showed that PERK was directly phosphorylated by AMPK on at least two conserved residues (serine 439 and threonine 680) and that AMPK activators elicited a PERK/eIF2A signaling cascade independent of the endoplasmic reticulum stress response in AML cells. CRISPR/Cas9 depletion and complementation assays illuminated a critical role for PERK in apoptotic cell death induced by pharmacological AMPK activation. Indeed, GSK621 induced mitochondrial membrane depolarization and apoptosis in AML cells, an effect that was mitigated when cells were depleted of PERK or expressed PERK with a loss of function AMPK phosphorylation site mutation. We identified the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) as a downstream target of the AMPK/PERK pathway, as its expression was enhanced in PERK knockdown AML cells. Moreover, selective pharmacologic activation of ALDH2 by the small molecule ALDA-1 recapitulated the protective effects of PERK depletion in the face of pharmacological AMPK activation. Corroborating the impact of the AMPK/PERK axis on mitochondrial apoptotic function, BH3 profiling showed marked Bcl-2 dependency in AML cells treated with GSK621. This dependency was abrogated in PERK-depleted cells, suggesting a role for PERK in mitochondrial priming to cell death. In vitro drug combination studies further demonstrated synergy between the clinical grade Bcl-2 inhibitor venetoclax (ABT-199) and each of four AMPK activators (GSK621, MK-8722, PF-06409577 and compound 991) in multiple AML cell lines. Finally, the addition of GSK621 to venetoclax enhanced anti-leukemic activity in primary AML patient samples ex vivo and in humanized mouse models in vivo. These findings together clarify the mechanisms of cytotoxicity induced by AMPK activation and suggest that combining pharmacologic AMPK activators with venetoclax may hold therapeutic promise in AML. References 1. Lin S-C, Hardie DG. AMPK: Sensing Glucose as well as Cellular Energy Status. Cell Metabolism. 2018;27(2):299-313. 2. Hardie DG. Molecular Pathways: Is AMPK a Friend or a Foe in Cancer? Clinical Cancer Research. 2015;21(17):3836-3840. 3. Jeon S-M, Hay N. The double-edged sword of AMPK signaling in cancer and its therapeutic implications. Arch. Pharm. Res. 2015;38(3):346-357. 4. Sujobert P, Poulain L, Paubelle E, et al. Co-activation of AMPK and mTORC1 Induces Cytotoxicity in Acute Myeloid Leukemia. Cell Rep. 2015;11(9):1446-1457. Figure Disclosures Tamburini: Novartis pharmaceutical: Research Funding; Incyte: Research Funding.

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