scholarly journals Immunosuppression and Outcomes in Acute Myeloid Leukemia

2021 ◽  
Author(s):  
Francesca Ferraro ◽  
Christopher Miller ◽  
Keegan Christensen ◽  
Nichole M. Helton ◽  
Margareth O'Laughlin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Rna Sequencing ◽  
Mhc Class Ii ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Independent Set ◽  
Normal Karyotype ◽  
Acute Myeloid

Acute myeloid leukemia (AML) patients rarely have long first remissions (> 5 years) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, short remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA sequencing and functional immunologic studies, we characterized 28 Normal Karyotype (NK)-AML patients with >5 year first remissions after chemotherapy (Long First Remissions, LFR) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 years (Standard First Remissions, SFR). Our combined analyses indicated that genetic risk profiling at presentation (as defined by ELN 2017 Criteria) was not sufficient to explain the outcomes of many SFR cases. Single cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T-cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells, or blocking the inhibitory MHC Class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ Tcell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

scholarly journals Immunosuppression and outcomes in adult patients with de novo acute myeloid leukemia with normal karyotypes

2021 ◽  
Vol 118 (49) ◽  
pp. e2116427118
Author(s):  
Francesca Ferraro ◽  
Christopher A. Miller ◽  
Keegan A. Christensen ◽  
Nichole M. Helton ◽  
Margaret O’Laughlin ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Rna Sequencing ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
De Novo ◽  
Independent Set ◽  
Normal Karyotype ◽  
Acute Myeloid

Acute myeloid leukemia (AML) patients rarely have long first remissions (LFRs; >5 y) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, standard remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing, and functional immunologic studies, we characterized 28 normal karyotype (NK)-AML patients with >5 y first remissions after chemotherapy (LFRs) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 y (standard first remissions [SFRs]). Our combined analyses indicated that genetic-risk profiling at presentation (as defined by European LeukemiaNet [ELN] 2017 criteria) was not sufficient to explain the outcomes of many SFR cases. Single-cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells or blocking the inhibitory major histocompatibility complex class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell–mediated suppression of CD4+ T cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.

scholarly journals A novel C2 domain binding CD33xCD3 bispecific antibody with potent T-cell redirection activity against acute myeloid leukemia

2020 ◽  
Vol 4 (5) ◽  
pp. 906-919 ◽  
Author(s):  
Priyanka Nair-Gupta ◽  
Michael Diem ◽  
Dara Reeves ◽  
Weirong Wang ◽  
Robert Schulingkamp ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
C2 Domain ◽  
Cytokine Release ◽  
Cynomolgus Monkeys ◽  
Acute Myeloid

Abstract CD33 is expressed in 90% of patients with acute myeloid leukemia (AML), and its extracellular portion consists of a V domain and a C2 domain. A recent study showed that a single nucleotide polymorphism (SNP), rs12459419 (C > T), results in the reduced expression of V domain–containing CD33 and limited efficacy of V domain–binding anti-CD33 antibodies. We developed JNJ-67571244, a novel human bispecific antibody capable of binding to the C2 domain of CD33 and to CD3, to induce T-cell recruitment and CD33+ tumor cell cytotoxicity independently of their SNP genotype status. JNJ-67571244 specifically binds to CD33-expressing target cells and induces cytotoxicity of CD33+ AML cell lines in vitro along with T-cell activation and cytokine release. JNJ-67571244 also exhibited statistically significant antitumor activity in vivo in established disseminated and subcutaneous mouse models of human AML. Furthermore, this antibody depletes CD33+ blasts in AML patient blood samples with concurrent T-cell activation. JNJ-67571244 also cross-reacts with cynomolgus monkey CD33 and CD3, and dosing of JNJ-67571244 in cynomolgus monkeys resulted in T-cell activation, transient cytokine release, and sustained reduction in CD33+ leukocyte populations. JNJ-67571244 was well tolerated in cynomolgus monkeys up to 30 mg/kg. Lastly, JNJ-67571244 mediated efficient cytotoxicity of cell lines and primary samples regardless of their SNP genotype status, suggesting a potential therapeutic benefit over other V-binding antibodies. JNJ-67571244 is currently in phase 1 clinical trials in patients with relapsed/refractory AML and high-risk myelodysplastic syndrome.

scholarly journals L4 Synthetic agonistic receptor-activating BiTEs – a modular platform for the efficient targeting of acute myeloid leukemia

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A2.2-A3
Author(s):  
M Benmebarek ◽  
BL Cadilha ◽  
M Hermann ◽  
S Lesch ◽  
C Augsburger ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Tumour Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Acute Myeloid

BackgroundTargeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). Due to the mutational landscape and heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target AML-associated antigen, and a cross-linking bispecific T cell engager (BiTE) specific for both (SAR) T cell and tumour cell.Materials and MethodsA SAR composed of an extracellular EGFRvIII, trans-membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap-extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific bispecific T cell engagers (BiTE) that target AML-associated antigens were designed and expressed in Expi293FTMcells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigen CD33.ResultsCD33-EGFRvIII BiTE, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by BiTE could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR.BiTE combination could also mediate specific cytotoxicity against patient-derived AML blasts whilst driving SAR T cell activation. In vivo, treatment with SAR.BiTE combination could efficiently eradicate leukemia and enhance survival in an AML xenograft model. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility of said activation upon depletion of the T cell engaging molecule.ConclusionsHere we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of agonistic receptor-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its BiTE facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells.Disclosure InformationM. Benmebarek: None. B.L. Cadilha: None. M. Hermann: None. S. Lesch: None. C. Augsburger: None. B. Brauchle: None. S. Stoiber: None. A. Darwich: None. F. Rataj: None. C. Klein: A. Employment (full or part-time); Significant; Roche. K. Hopfner: None. M. Subklewe: None. S. Endres: None. S. Kobold: None.

scholarly journals Bifunctional PD-1 × αCD3 × αCD33 fusion protein reverses adaptive immune escape in acute myeloid leukemia

Blood ◽  
2018 ◽  
Vol 132 (23) ◽  
pp. 2484-2494 ◽  
Author(s):  
Monika Herrmann ◽  
Christina Krupka ◽  
Katrin Deiser ◽  
Bettina Brauchle ◽  
Anetta Marcinek ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Adaptive Immune ◽  
Acute Myeloid

Abstract The CD33-targeting bispecific T-cell engager (BiTE) AMG 330 proved to be highly efficient in mediating cytolysis of acute myeloid leukemia (AML) cells in vitro and in mouse models. Yet, T-cell activation is correlated with upregulation of programmed cell death-ligand 1 (PD-L1) and other inhibitory checkpoints on AML cells that confer adaptive immune resistance. PD-1 and PD-L1 blocking agents may counteract T-cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a bifunctional checkpoint inhibitory T cell–engaging (CiTE) antibody that combines T-cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade. This is accomplished by fusing the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1, to an αCD3.αCD33 BiTE-like scaffold. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increases T-cell activation (3.3-fold elevation of interferon-γ) and leads to efficient and highly selective cytotoxicity against CD33+PD-L1+ cell lines (50% effective concentration = 2.3-26.9 pM) as well as patient-derived AML cells (n = 8). In a murine xenograft model, the CiTE induces complete AML eradication without initial signs of irAEs as measured by body weight loss. We conclude that our molecule preferentially targets AML cells, whereas high-affinity blockers, such as clinically approved anticancer agents, also address PD-L1+ non-AML cells. By combining the high efficacy of T-cell engagers with immune checkpoint blockade in a single molecule, we expect to minimize irAEs associated with the systemic application of immune checkpoint inhibitors and suggest high therapeutic potential, particularly for patients with relapsed/ refractory AML.

scholarly journals Microenvironment Produced by Acute Myeloid Leukemia Cells Prevents T Cell Activation and Proliferation by Inhibition of NF-κB, c-Myc, and pRb Pathways

2001 ◽  
Vol 167 (10) ◽  
pp. 6021-6030 ◽  
Author(s):  
Andrea G. S. Buggins ◽  
Dragana Milojkovic ◽  
Matthew J. Arno ◽  
Nicholas C. Lea ◽  
Ghulam J. Mufti ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Leukemia Cells ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

scholarly journals Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2171
Author(s):  
Isabel Valhondo ◽  
Fakhri Hassouneh ◽  
Nelson Lopez-Sejas ◽  
Alejandra Pera ◽  
Beatriz Sanchez-Correa ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Healthy Volunteers ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

RNA-Sequencing Unveils Cryptic Fusions in Patients with Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1278-1278
Author(s):  
Fabiana Ostronoff ◽  
Matthew Fitzgibbon ◽  
Martin McIntosh ◽  
Rhonda E. Ries ◽  
Alan S. Gamis ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Prognostic Markers ◽  
Normal Karyotype ◽  
Fusion Transcript ◽  
Rna Seq ◽  
Illumina Hiseq ◽  
Normal Controls ◽  
Acute Myeloid

Abstract Abstract 1278 Introduction: Acute myeloid leukemia (AML) represents a heterogeneous group of malignancies with great variability in response to therapy. In recent years, an increasing list of molecular markers with prognostic significance in AML has been identified; nonetheless, new prognostic markers and therapeutic targets are still needed. The aim of this study was to identify and verify fusion transcripts using RNA-Sequencing (RNA-Seq) that would be otherwise undetectable by conventional karyotyping. Methods: Transcriptome Sequence data is generated by high-throughput short-read RNA-Seq performed for each AML sample on the Illumina HiSeq. Poly(A) RNA is captured with poly(T) magnetic beads, fragmented, copied to cDNA libraries with reverse transcriptase and random primers. Each library is subjected to 50-cycle paired-end sequencing on the Illumina HiSeq at Hudson Alpha. Filtered Fastq files are processed with TopHat-Fusion [Kim2011,Trapnell2009] alignment software to discover cryptic fusions in RNA-Seq data without relying on known, annotated models. This process yielded an average of 20 million alignable reads per sample. Cord blood blast cell transcripts are also processed and serve as normal controls. A series of filtering steps eliminate junctions commonly found to be in error. Filtered junctions found in at least 3 AML samples and no normal controls are retained as AML-associated candidate junctions. Visual curation of candidates is performed using Integrative Genomics Viewer. Candidate fusions were verified by RT-PCR amplification of the AML-associated fusions in the index cases. Fusion transcript product, as well as the break point junction was verified by Sanger sequencing Results: Diagnostic specimens from 70 patients with de novo AML that included patients with normal karyotype (NK, N=31), core-binding factor (CBF) AML (N=33) and other (N=6) were sequenced. Age at diagnosis varied from 10 months to 69 years (Median 12 years). White blood cell count (WBC) and blast percentage were 49×109/L (range, 2.4 to 496×109/L) and 78% (40% to 100%), respectively. Bioinformatic evaluation of the RNA-Seq data revealed 67 high-value novel fusions that were not detected by conventional karyotyping: 54 (80.6%) were intra- and 13 (19.4%) inter-chromosomal junctions. The number of novel translocations varied in different cytogenetic groups, with 22 novel fusions detected in those with NK (16 intra and 6 inter-chromosomal junctions), 37 CBF (32 intra and 5 inter-chromosomal junctions) and 8 in “other” (6 intra and 2 inter-chromosomal junctions). Thirteen novel fusions (19.4%) were found in at least 2 or more screened-patients: two (15.4%) inter- and 11 (84.6%) intra-chromosomal junctions. Median number of fusions identified per patient was 2 (range, 1 to 6). Novel fusions involving PDGFR-β gene were identified in two patients, each with a different translocation partner (G3BP1 and ETV6, which was an intra and inter-chromosomal fusions, respectively). Sequencing of the fusion transcript junctions verified the fusion junctions and demonstrated in frame fusions of G3BP1 and ETV6 to the kinase domain coding region of PDGFR-β, identical junction to that seen in cases of imatinib sensitive idiopathic hypereosinophilic syndrome (IHES). Frequency validation in 100 adult and 100 pediatric cases identified one additional patient with G3BP1-PDGFR-β. Cryptic NUP98/NSD1 was identified and verified in two patients with normal karyotype as well as NUP98/HOXD13 translocation in one patient. Frequency determination of NUP98/NSD1 demonstrated prevalence of 7.8% in patients with NK, and that of 13% in patients with FLT3/ITD. Patients who harbored both NUP98/NSD1 fusion and FLT3/ITD had a dismal remission induction rate (CR rate in FLT3/ITD with and without NUP98/NSD1 was 28% vs. 73%; p=0.002). Conclusion: Our data show the applicability of RNA-Seq as a tool to discover cryptic fusion transcripts in AML. These novel fusions may define new independent prognostic markers and potential therapeutic targets for patients with this highly treatment-resistant disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Drug-DNA Conjugated Gold Nanoparticles for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4935-4935
Author(s):  
Nathan Gossai ◽  
Jordan Naumann ◽  
Ed Zamora ◽  
Nan-Sheng Li ◽  
Joseph Piccirilli ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Acute Myeloid Leukemia ◽  
T Cell Activation ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Cd34 Cells ◽  
Acute Myeloid ◽  
Novel Therapeutic

Abstract INTRODUCTION: Novel therapies are needed for acute myeloid leukemia as only ~60% of children are cured despite maximally intensive cytotoxic chemotherapy. Functionalized gold nanoparticles (AuNP) are utilized for many biomedical applications and represent a potentially novel therapeutic approach in leukemia. We enhanced this technology by developing an AuNP system that selectively releases molecularly targeted drugs in leukemia cells. METHODS: AuNPs were functionalized with short, double-stranded oligonucleotides with sequence complementarity to genes overexpressed in or unique to a leukemia cell (e.g. survivin or AML/ETO). Only the anti-sense oligonucleotide is covalently bound to the nanoparticle via a thiol linker. Thus, after entering leukemia cells, the endogenous targeted oncogene mRNA can bind to its complementary sequence on the AuNP and displace the non-covalently bound oligonucleotide which, in our system, is conjugated to the multi-tyrosine kinase inhibitor dasatinib. As a binary reaction, the amount of dasatinib-conjugated oligonucleotide released from the nanoparticle is directly proportional to both the presence and abundance of the complementary mRNA present in a cell. We evaluated AuNP uptake into multiple AML cell lines, as well as normal hematopoietic cells. The effect of dasatinib-AuNPs on dasatinib-sensitive leukemia cell lines was evaluated using proliferation assays, annexin V staining, and cell colony assays. Toxicity in T-cells and CD34+ cells was assessed with T-cell activation and p-SRC assays, respectively. RESULTS: Conjugation of dasatinib to an oligonucleotide complementary to a region of the survivin gene did not perturb its ability to inhibit SRC and c-KIT kinases in vitro. Leukemia cells demonstrate highly efficient AuNP uptake when cultured alone or with up to a 100-1000 fold excess of normal bone marrow cells. Treatment of K562 leukemia cells, containing a BCR/ABL translocation and high levels of survivin mRNA, with dasatinib-AuNPs resulted in dose-dependent p-SRC and p-CRKL inhibition. Furthermore, K562 cells also showed significantly impaired proliferation, increased apoptosis, and formed fewer colonies in methylcellulose. Conversely, normal T-cells and CD34+ cells, which express less survivin than leukemia cells, were significantly less affected by dasatinib-AuNPs than dasatinib alone as measured by T-cell activation assays and p-SRC levels, respectively. CONCLUSIONS: This method of using functionalized AuNPs to deliver and activate dasatinib in leukemia cells augments drug efficacy while minimizing toxicity and thus represents a novel therapeutic strategy for AML. Ongoing work is underway to characterize the mechanism of preferential AuNP uptake in leukemia cells and assessing in vivo activity using a murine xenotransplantation model of leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mutation detection in thousands of acute myeloid leukemia cells using single cell RNA-sequencing

2018 ◽  
Author(s):  
Allegra A. Petti ◽  
Stephen R. Williams ◽  
Christopher A. Miller ◽  
Ian T. Fiddes ◽  
Sridhar N. Srivatsan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Single Cell ◽  
Rna Sequencing ◽  
Myeloid Leukemia ◽  
Normal Karyotype ◽  
Integrative Approach ◽  
Single Cell Rna Sequencing ◽  
Acute Myeloid

AbstractVirtually all tumors are genetically heterogeneous, containing subclonal populations of cells that are defined by distinct mutations1. Subclones can have unique phenotypes that influence disease progression2, but these phenotypes are difficult to characterize: subclones usually cannot be physically purified, and bulk gene expression measurements obscure interclonal differences. Single-cell RNA-sequencing has revealed transcriptional heterogeneity within a variety of tumor types, but it is unclear how this expression heterogeneity relates to subclonal genetic events – for example, whether particular expression clusters correspond to mutationally defined subclones3,4,5,6-9. To address this question, we developed an approach that integrates enhanced whole genome sequencing (eWGS) with the 10x Genomics Chromium Single Cell 5’ Gene Expression workflow (scRNA-seq) to directly link expressed mutations with transcriptional profiles at single cell resolution. Using bone marrow samples from five cases of primary human Acute Myeloid Leukemia (AML), we generated WGS and scRNA-seq data for each case. Duplicate single cell libraries representing a median of 20,474 cells per case were generated from the bone marrow of each patient. Although the libraries were 5’ biased, we detected expressed mutations in cDNAs at distances up to 10 kbp from the 5’ ends of well-expressed genes, allowing us to identify hundreds to thousands of cells with AML-specific somatic mutations in every case. This data made it possible to distinguish AML cells (including normal-karyotype AML cells) from surrounding normal cells, to study tumor differentiation and intratumoral expression heterogeneity, to identify expression signatures associated with subclonal mutations, and to find cell surface markers that could be used to purify subclones for further study. The data also revealed transcriptional heterogeneity that occurred independently of subclonal mutations, suggesting that additional factors drive epigenetic heterogeneity. This integrative approach for connecting genotype to phenotype in AML cells is broadly applicable for analysis of any sample that is phenotypically and genetically heterogeneous.

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