All Roads Lead to Rome: Phenotypic Channeling to Uniform Differentiation Structures from Diverse Leukemia Genotypes by Mass Cytometry

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-22-SCI-22
Author(s):  
Garry Nolan
Keyword(s):  
Stem Cells ◽  
B Cell ◽  
Single Cell ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
Signaling Networks ◽  
Mass Cytometry ◽  
Human Cord Blood

Abstract Emerging single-cell technologies have been pivotal in uncovering an extensive degree of heterogeneity between and within tissues (1). Analysis of single-cell data has shed light on many different cellular processes (2-7) and recent technological advances have enabled the study of a large number of parameters in single cells at unparalleled resolution. One such technology, mass cytometry (8), can measure up to 45 parameters simultaneously in tens of thousands of individual cells. Using mass cytometry and genomic sequencing of conventionally sorted subpopulations show that acute myelogenous leukemia (AML) in a given patient can simultaneously occupy multiple stages of differentiation. Occupation of these stages was correlated with the presence, or absence, of unique exonic mutation fingerprints. In another cancer, B-cell acute lymphoblastic leukemia (ALL), outgrowth of tumor at pro and pre-B cell stages was nearly always uniquely at a single stage - contrary to the results in AML. This suggests that evolutionary “niche” searching is not only for physical space in cancers, but also involves utilization of differentiation machinery as an additional elaboration mechanism. Each differentiation stage in both AML and B-cell ALL was characterized by utilization of cognate signaling networks which showed differential susceptibility to drug action. Using such deep profiling and signaling delineation approaches at the single-cell level will allow for fine structured indexing of patient disease and further tailoring of disease management. In addition, it will allow “heterogeneous” tumors to be organized by a maturation index associated with a granular catalog of mutations that drive cells to occupy these pseudo-differentiation niches. 1. Bendall, S.C., et al., A deep profiler's guide to cytometry.Trends Immunol, 2012. 33(7): p. 323-32. 2. Petilla Interneuron Nomenclature Group, et al., Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.Nat Rev Neurosci, 2008. 9(7): p. 557-68. 3. Irish, J.M., et al., Single cell profiling of potentiated phospho-protein networks in cancer cells.Cell, 2004. 118(2): p. 217-28. 4. Sachs, K., et al., Causal protein-signaling networks derived from multiparameter single-cell data.Science, 2005. 308(5721): p. 523-9. 5. Majeti, R., C.Y. Park, and I.L. Weissman, Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell, 2007. 1(6): p. 635-45. 6. Tarnok, A., H. Ulrich, and J. Bocsi, Phenotypes of stem cells from diverse origin.Cytometry A, 2010. 77(1): p. 6-10. 7. O'Brien, C.A., A. Kreso, and J.E. Dick, Cancer stem cells in solid tumors: an overview.Semin Radiat Oncol, 2009. 19(2): p. 71-7. 8. Bandura, D.R., et al., Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem, 2009. 81(16): p. 6813-22. Disclosures: No relevant conflicts of interest to declare.

scholarly journals McSNAC: A software to approximate first-order signaling networks from mass cytometry data

2021 ◽  
Author(s):  
Darren Wethington ◽  
Sayak Mukherjee ◽  
Jayajit Das
Keyword(s):  
Single Cell ◽  
In Silico ◽  
Single Cells ◽  
Signaling Network ◽  
Signaling Networks ◽  
Enzymatic Reactions ◽  
Signaling Proteins ◽  
Protein Species ◽  
Mass Cytometry ◽  
First Order

AbstractMass cytometry (CyTOF) gives unprecedented opportunity to simultaneously measure up to 40 proteins in single cells, with a theoretical potential to reach 100 proteins. This high-dimensional single-cell information can be very useful to dissecting mechanisms of cellular activity. In particular, measuring abundances of signaling proteins like phospho-proteins can provide detailed information on the dynamics of single-cell signaling processes. With a proper computational analysis, timestamped CyTOF data of signaling proteins could help develop predictive and mechanistic models for signaling kinetics. These models would be useful for predicting the effects of perturbations in cells, or comparing signaling networks across cell groups. We propose our Mass cytometry Signaling Network Analysis Code, or McSNAC, a new software capable of reconstructing signaling networks and estimating their kinetic parameters from CyTOF data.McSNAC approximates signaling networks as a network of first-order reactions between proteins. This assumption breaks down often as signaling reactions can involve binding and unbinding, enzymatic reactions, and other nonlinear constructions. Furthermore, McSNAC may be limited to approximating indirect interactions between protein species, as cytometry experiments are only able to assay a small fraction of the protein species that are involved in signaling. We carry out a series of in silico experiments here to show that 1) McSNAC is capable of accurately estimating the ground-truth model in a scalable manner when given data originating from a first-order system; 2) McSNAC is capable of qualitatively predicting outcomes to perturbations of species abundances in simple second-order reaction models and in a complex in silico nonlinear signaling network in which some proteins are unmeasured. These findings demonstrate that McSNAC can be a valuable screening tool for generating models of signaling networks from timestamped CyTOF data.

Single-Cell Phospho-Profiling in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia Reveals Signaling Differences in Cytogenetic and Prognostic Subgroups.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2653-2653
Author(s):  
Manon Queudeville ◽  
Sarah M. Eckhoff ◽  
Klaus-Michael Debatin ◽  
Lueder H. Meyer
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Single Cell ◽  
Poor Prognosis ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Cytogenetic Aberrations

Abstract Abstract 2653 Poster Board II-629 Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in combination with surface staining in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level. We anayzed 22 xenograft samples derived from NOD/SCID-mice transplanted with primary pediatric B- cell precursor acute lymphoblastic leukemia (BCP- ALL) cells. The cells were isolated from the spleens of leukemia bearing mice and stimulated ex vivo in vitro with different stimulants and cytokines. Activation of various phosphoepitopes was analyzed by flow cytometry and compared to the basal state of unstimulated samples. TEL/AML1 fusion and MLL-rearrangements are the most common cytogenetic aberrations in childhood BCP- ALL and are associated with a good or very poor prognosis, respectively. Although there were no differences detectable in basal phosphorylation between the different cytogenetic subgroups, TEL/AML1- positive samples (n= 5) displayed a significantly lower phosphorylation of extracellular regulated kinase (ERK1/2) after stimulation with PMA (Phorbol-12-myristat-13-acetate, activator of protein kinase C) or interleukin 7 (IL-7), while they showed a significantly higher activation of p38 after stimulation with PMA, compared to samples without translocation (n= 13). Additionally, the fusion gene negative samples showed a downregulation of STAT1-phosphorylation after stimulation with interleukin 10 (IL-10) whereas the TEL/AML1-positive samples showed no change. Interestingly, the MLL- positive samples (n= 3) also did not show a difference in STAT1-phosphorylation after IL-10, but showed significantly stronger STAT1 activation in response to interferon alpha (IFN-a) compared to samples without fusion genes. Moreover, the MLL- positive samples also displayed a weaker reaction in ERK-phosphorylation after IL-7 compared to the leukemia samples without cytogenetic aberrations. Differences in other prognostic subgroups analysed include a weaker phosphorylation of p38 and JNK after anisomycin in samples where the patient initially presented with hyperleucocytosis (> 100.000 WBC/μl) (n= 3), an indicator of poor prognosis. A decrease in STAT3- activation after IL-10 was observed in samples where the patients displayed bone marrow remission on day 15 of therapy (n= 8), compared to no change in the samples of patients with > 5% residual blasts (n= 8), indicative of therapy resistance, at this timepoint. Similar to the results for the cytogenetic subgroups, there were no differences detectable at basal phosphorylation levels between the prognostic subgroups. Taken together, these data show that basal phosphorylation states of specific signaling molecules do not discriminate between the different prognostic subgroups of BCP- ALL analyzed. Cytogenetic and other prognostic subgroups however display specific profiles of signaling networks after stimulation. This strategy will prove helpful to identify mechanisms by which different subgroups with distinct clinical outcomes interpret environmental signals and hereby define pathways important for continued survival, proliferation and resistance eventually leading to novel biomarkers and targeted therapies. Disclosures: No relevant conflicts of interest to declare.

Identification of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment and Self-Renewal.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 816-816
Author(s):  
Faiyaz Notta ◽  
Sergei Doulatov ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Insight Into

Abstract Abstract 816 A fundamental tenet that has guided our insight into the biology of hematopoietic stem cells (HSCs) over the past 50 years is the principle that an HSC can only be assayed by functional repopulation of an irradiated host1. In its strictest definition, only a HSC can provide long-term reconstitution of all the major lineages following single cell transplantation. However, the existing strategies for human HSC isolation lack quantitation and do not submit to this rigorous standard, thus precluding further biological analysis. Here, we report the prospective and quantitative analysis of human cord blood (CB) HSCs transplanted into female NOD/SCID/IL-2Rgcnull mice. We identify integrin a6 (CD49f) as a novel marker of cord blood (CB) HSCs and report that single Lin-CD34+CD38-CD90+CD45RA-RholoCD49fhi cells can reconstitute myeloid, B-, and T-cell lineages for 18 weeks. 5 of 29 mice transplanted with single cells gave rise to human cells indicating that approximately 20% of cells in this fraction are HSCs. This advance finally enables utilization of near-homogeneous populations of human HSCs to gain insight into their biology and to harness them for stem cell-based therapeutics. Disclosures: No relevant conflicts of interest to declare.

Mass Cytometry Organizes the Heterogeneity of Pediatric B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 753-753
Author(s):  
Kara L. Davis ◽  
Erin F. Simonds ◽  
Sean C Bendall ◽  
Wendy J. Fantl ◽  
Garry P. Nolan
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Single Cell ◽  
Lymphoblastic Leukemia ◽  
Patient Specific ◽  
Phenotypic Traits ◽  
Surface Markers ◽  
Mass Cytometry ◽  
Variable Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 753 Pediatric B Cell Acute Lymphoblastic Leukemia: Common and Unique Differentiation States Defined by Signaling Response States Background: When mutations or regulatory dysfunction drive inappropriate cell division or survival this sets the stage for cancer initiation or progression. At what point do cells in an early cancer veer from their normal cellular routines to no longer participate in the development of a normal cellular tissue architecture & differentiation heirarchy? Do they still attempt to play out their programming to some degree, or are they “free actors”? The fact that cancers of any given type appear to remain tied to phenotypic classification schemes is illustrated in traditional clinical stratification systems. Paradoxically, cancers are considered (by some) as “heterogeneous”, whereas as a class they appear to recapitulate patterns of clinical responses, gene expression, signaling states. To what extent are these repeated molecular patterns mirrored at the level of differentiation? We mapped, at the single cell level, patterns of expression of markers and phenotypic traits that might be similar or unique across patient subgroups of pediatric B-cell ALL samples using 35 parameter proteomic mass cytometry. Using the features assignable to each single cell, with a statistical reconstruction of most likely similarity of features in 35 dimensional space, we mapped a “common” differentiation tree previously unrecognized by conventional analyses, and demonstrate here how differences in certain markers distinguish, or create, common phenotypic co-expression features across these ALL patients, or suggest patient-specific derailing of differentiation associated with changes in signaling module expression states. Methods: Cryopreserved cells were obtained from 8 pediatric B cell ALL patients under informed consent. 19 surface markers were used to cluster data into metacells of localized similarity displayed in a tree of local relationships via hierarchical cell lineage clustering and spanning-tree progression analysis of density-normalized events (SPADE) (Figure 1) (Bendall et al, Science 6 May2011; 332: 687–696). Results: The blast cell subpopulations comprised the areas of greatest density on the spanning trees–consistent with the fact that these cells are the most prevalent. Blast populations displayed variable expression of B cell precursor surface markers such as CD10, CD19, CD34 and CD38—even though clinical phenotyping placed all patients in a similar class. SPADE analysis detailed unexpected sub-branches prominently observed in certain patients, while absent or weakly represented in others. This confirms common signaling and differentiation states can be observed across patients, but individual patients can manifest unique and prevalent outgrowths of these common malignant differentiation states. Three patients' disease appears to gain a transformative event during a common point at a “pre-B cell development state, leading to local expansion at this halted population of characteristic immunophenotype. Sample ALL04 appears to have an outgrowth of cells with the earliest pre-B cell progenitors, whereas ALL01 is consistent with a maturing pre B cell, gaining expression of CD45. Notably, ALL03, characterized by an MLL rearrangement, clusters with myeloid cells and is CD10 negative, suggesting transformation prior to lymphocyte commitment. The cellular responses to perturbation provides added structure to the cellular subsets; responses to stimulation of the preB cell receptor are present within the majority of blast populations for ALL01 but absent in most for ALL03 and ALL04. We will present this and other findings related to these patients, as well as application of this approach to other tumor types. Conclusion:This high-dimensional immunophenotypic analysis of single cells from primary patient samples reveals an unseen developmental structure within pediatric B precursor acute lymphoblastic leukemia. The developmental stage at which the transformative event occurs informs the characteristic response to perturbation and critically, to drug treatment. *KD and ES contributed equally to this work. Disclosures: Fantl: Nodality, Inc.: Equity Ownership.

scholarly journals Leukemia Stem Cells in Acute Lymphoblastic Leukemia: Unveiling Hierarchical Structure at Single Cell Resolution

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4786-4786
Author(s):  
Noriko Satake ◽  
Connie Duong ◽  
Sakiko Yoshida ◽  
Ryan Davis ◽  
Stephenie Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
B Cell ◽  
Single Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Type ◽  
Transcriptome Profile ◽  
Nsg Mice ◽  
Novel Method

Abstract Leukemia stem cells (LSCs) are the root of leukemia, and are responsible for drug resistance and disease relapse. However, LSCs have not yet been identified for acute lymphoblastic leukemia (ALL). Among many challenges, lack of phenotypic markers is one of the major problems in identifying ALL LSCs. In this study, we demonstrated a novel method to isolate LSCs from both T- and B- cell ALLs and further characterized their transcriptome profile at the single cell level. We have recently identified a novel method to isolate ALL LSCs based on cellular metabolic activities. We demonstrated that these isolated LSCs had in vivo leukemia-initiating capability (LIC). We have developed a series of primary ALL xenograft mouse models using patient samples and NOD/SCID/IL2Rg-/- (NSG) mice. Leukemia cells harvested from several generations of these mice were used in this study. We isolated LSCs and non-LSCs from 4 different B-cell type ALL samples and transplanted them separately into healthy NSG mice. Cell numbers used varied between 5, 10, and 50,000 per mouse, and the number of the animals varied between three and eight per group. All the animals transplanted with LSCs developed leukemia between 5-14 weeks, whereas those transplanted with non-LSCs did not develop the disease within the same timeframe or by the end of the study, which was more than 4 months after leukemia development in the LSC group. In order to characterize and identify potential therapeutic targets in the LSCs, we investigated the transcriptome profile of these cells. First, we performed genomewide microarray gene expression profiling of RNA isolated from the LSCs and non-LSCs using 4 ALL cell lines (Reh, JM1, Jurkat, and Molt4). There were 173 genes which showed at least 2-fold difference in gene expression between the LSCs and non-LSCs. Using a panel of primer sets for the 100 genes exhibiting the highest difference in expression, we performed qRT-PCR for these genes in the isolated LSCs and non-LSCs from 11 primary ALL samples (10 B-cell and 1 T-cell type) transplanted and harvested from our NSG xenograft mouse models at different generations. There was a distinct difference in the transcriptome profile between the LSCs and non-LSCs in these primary ALL samples. Overall gene expression of 93 LSC signature genes was much lower in the LSCs than in the non-LSCs. Recent advances in microfluidic technologies allowed us to investigate cells at single cell resolution. Growing evidence suggests that cancer stem cells consist of heterogeneous cell populations (subclones). Therefore, we further investigated whether these isolated LSCs have subclones using the Fluidigm C1 and Biomark system. Preliminary results using a primary ALL sample harvested from our xenograft mouse model, indicate that there are at least two distinct subclones in the LSCs based on principal component analysis of the single cell data. In summary, we 1) developed a novel method to isolate ALL LSCs which have in vivo LIC, 2) demonstrated that isolated LSCs have a distinct transcriptome profile, and 3) discovered that the LSCs seem to consist of subclones. Currently we are in the process of performing detailed comprehensive transcriptome analyses and additional single cell transcriptome assays. Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemia-Initiating Activity in T-ALL Requires Active Hif1a and Wnt Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 57-57
Author(s):  
Vincenzo Giambra ◽  
Catherine E Jenkins ◽  
Sonya H Lam ◽  
Catherine Hoofd ◽  
Miriam Belmonte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Negative Regulator ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Hematopoietic Stem

Abstract Prior work has shown that NOTCH1 is a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL) with activating NOTCH1 mutations occurring in over 50% of cases (Weng et al, Science 2004) and loss-of-function mutations in its negative regulator FBXW7 occurring in 8-15% of cases (O’Neil et al, J Exp Med 2007; Thompson et al, J Exp Med 2007). Subsequent work has shown that continued Notch signaling is required for maintenance of T-ALL leukemia stem cells (Armstrong et al, Blood 2009; Tatarek et al, Blood 2011; Giambra et al, Nat Med 2012). Several lines of evidence have substantiated genetic interactions between the Notch and Wnt signaling pathways in various contexts, and Wnt signaling has been shown to play important roles in hematopoietic stem cell biology and also in hematopoietic cancers such as acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Luis et al, Leukemia 2012). To address what role if any Wnt signaling may play in T-ALL, we generated primary murine leukemias by viral transduction of bone marrow progenitors with activated NOTCH1, then delivered a fluorescent Wnt reporter construct (7TGP; Fuerer & Nusse, PLoS ONE 2010) by lentiviral transduction, and retransplanted the leukemias to interrogate Wnt signaling activity in vivo. We report here that active Wnt signaling is restricted to minor subpopulations within bulk T-ALL tumors, and that these Wnt-active subsets are highly enriched for leukemia-initiating cell (LIC) activity. Moreover, using Ctnnb1loxP/loxP animals we show that inducible Cre-mediated deletion of β-catenin or enforced expression of a dominant-negative TCF construct severely compromises LIC activity. We also show that β-catenin levels are upregulated by hypoxia through Hif1a stabilization, and that deletion of Hif1a also severely compromises LIC activity. Interestingly, Wnt-active subsets are distributed diffusely throughout the marrow interstitial space suggesting that tumor infiltration induces formation of local hypoxic niches as opposed to taking up residence in pre-existing anatomic compartments with low oxygen tensions. Taken together, these results suggest a model in which hypoxic niches in vivo facilitate Hif1a-dependent accumulation of β-catenin which drives Wnt signaling and self-renewal of leukemia stem cells. Finally, we show using patient-derived xenografts that antagonism of Hif1a or Wnt signaling also compromises human LIC activity, suggesting that pharmacologic targeting of these pathways could have therapeutic application in patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

Mass Cytometry Analysis Dissects CRLF2-Driven Signaling Pathways in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 906-906
Author(s):  
Jolanda Sarno ◽  
Kara L. Davis ◽  
Angela Maria Savino ◽  
Cristina Bugarin ◽  
Stefania Pinto ◽  
...  
Keyword(s):  
Single Cell ◽  
Signaling Pathways ◽  
Cross Talk ◽  
Research Funding ◽  
Ex Vivo ◽  
Single Cells ◽  
Lymphoblastic Leukemia ◽  
Cell Mass ◽  
Control Group ◽  
Mass Cytometry

Abstract BACKGROUND: Rearrangements of the CRLF2 gene, present in 7-15% of childhood BCP-ALL, are responsible of the overexpression of Thymic Stromal Lymphopoietin Receptor (TSLPR) and they are correlated with poor prognosis (Chen IM Blood 2012). TSLPR overexpression can be associated with JAK2 mutations, which leads to aberrant activation of JAK/STAT and PI3K/AKT pathways. Although the cross talk of the signaling pathways is still under investigation, there is a rationale for the use of targeted tyrosine kinase inhibitors (TKIs) to treat this subgroup of patients (Maude SL Blood 2012). We focused on the dissection of CRLF2-driven signaling in primary CRLF2 rearranged(r) BCP-ALL samples by using single cell mass cytometry (CyTOF) analysis. We leveraged the high dimensional single cell capability of the CyTOF to understand, with previously unattainable resolution, the activation of these pathways simultaneously in single cells and their response to inhibition with TKIs and anti-TSLPR monoclonal antibodies (mAbs). This revealed heterogeneity in signaling response, identifying subpopulations which differentially activate intracellular signals through TSLPR and differentially respond to ex vivo treatment. METHODS: Twelve BCP-ALL primary samples, 6 CRLF2r and 6 CRLF2 wild type (wt), were investigated and the expression of 24 phenotypic and 15 functional proteins were measured at single cell level using CyTOF as previous described (Bendall SC Science 2011). To assess the response to ex-vivo TSLP stimulation (10ng/mL) and TKIs/mAbs treatment, data were normalized to the basal levels of each phosphoprotein and significance was calculated using student`s t-test. One million cells per condition were treated with different TKIs, Dasatinib, Ruxolitinib and BEZ-235, and two different clones of anti-TSLPR mAbs (130A10 and 130H3) from MRC Technology. RESULTS: As expected, we observed an aberrant TSLP-induced activation of pSTAT5 and prpS6 in CRLF2r patients as compared with CRLF2wt, used as control group (p=0.0055, p= 0.0007). Of note, we also observed a previously not described TSLP-dependent activation of pERK and pCREB (p=0.0313, p=0.0261) suggesting a cross-talk of the TSLPR-driven signaling also with the RAS/MEK pathway. Treatment with TKIs revealed strong inhibitory activity of Dasatinib, which completely inhibited the TSLP-mediated phosphorylation of STAT5, rpS6, CREB and ERK in CRLF2r treated blasts compared to CRLF2r not treated cells (p= 0.0040, 0.0017, 0.0007, 0.0114 respectively). Ruxolitinib, JAK1/2 inhibitor, also reduced rpS6, CREB and ERK phosphorylation (p=0.0025, 0.037, 0.0132). Interestingly one of the two anti-TSLPR tested mAbs (130A10) was also able to significantly inhibit the TSLP-mediated activation of STAT5, rpS6, and ERK (p= 0.0071, 0.0006, 0.0323). Finally, the PI3K/TORk inhibitor, BEZ-235, did not show any statistically significant reductions. Single cell analysis revealed a population of TSLPR overexpressing blasts (range 20-50%) in which the TSLP stimulation resulted in activation of prpS6 but not pSTAT5, present in all the CRLF2r patients. This rpS6 activation could be inhibited by anti-TSLPR mAb, Dasatinib, Ruxolitinib and BEZ-235, except for one patient in which the activation was blunted only by anti-TSLPR mAb and Dasatinib suggesting an activation of prpS6 through a non canonical pathway. This data reveals heterogeneous signaling populations present within this subtype of leukemia driven by TSLPR overexpression. Finally in 3 additional CRLF2r primary samples, we investigated signaling profile of residual blasts (MRD) at Day8 and Day15 post induction initiation. TSLPR expression was consistently maintained in all patients at both time points. Furthermore, residual blasts were still able to respond to TSLP and the induced pSTAT5 could be effectively inhibited by 130A10 anti-TSLPR clone and Ruxolitinib. CONCLUSION: In summary, these data suggest heterogeneity of TSLPR-related signaling with activation of the expected JAK/STAT and PI3K pathways but also RAS/MEK and CREB activation. Further, TSLPR+ blasts exhibit heterogeneous responses to both treatment with TSLP in combination with TKIs or mAb. Finally, the MRD detection by CyTOF allowed the study of the functional activity of the TSLPR positive resistant cells suggesting a role of CRLF2r in the persistence of the leukemic cells and its targeting to treat late and refractory stages of the disease. Disclosures Davis: Fluidigm, Inc: Honoraria. Dyer:Roche Pharmaceuticals: Speakers Bureau; Gilead: Research Funding; ONO Pharmaceuticals: Research Funding. Nolan:Fluidigm, Inc: Equity Ownership.

BCR/ABL Can Promote CD19+ Cell Growth but Not Render Them Long-Term Stemness

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2709-2709
Author(s):  
Donghe Li ◽  
Xuemei Zhao ◽  
Bo Jiao ◽  
Ping Liu ◽  
Ruibao Ren
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
T Cell Therapy ◽  
Hematopoietic Stem

Abstract Cancer stem cells are a subpopulation of malignant cells that have the capacity of both self-renewal and reconstitution of the cancer. Eradication of cancer stem cells is crucial for curing the malignant disease. Previous studies in hematopoietic malignancies showed that leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) chronic phase are originated from a hematopoietic stem cell (HSC), while LSCs in acute myeloid leukemia (AML) can either be derived from HSCs or be transformed from myeloid progenitors. But in acute B-lymphoblastic leukemia (B-ALL), the origin of leukemia stem cells is not clear. In this study, we tested whether BCR/ABL could transform B-lineage committed CD19+ cells to LSCs. We found that transducing BCR/ABL in CD19+ cells can promote their colony formation in vitro and induce B-ALL like disease in vivo. However, only BCR/ABL transduced whole bone marrow cells, but not CD19+ cells, can be transplanted multiple times in recipient mice, and the frequency of long-term LSCs from the latter ranges from 1/135 to 1/629. These studies suggest that LSCs in BCR/ABL+ B-ALL may originate from CD19- hematopoietic stem/progenitor cells and that CD19 chimeric antigen receptor (CAR) modified T cell therapy may not be effective in eradicating LSCs in BCR/ABL+ B-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell Developmental Classification of B-Cell Precursor Acute Lymphoblastic Leukemia at Diagnosis Reveals Predictors of Relapse

2018 ◽  
Vol 64 ◽  
pp. S33-S34 ◽  
Author(s):  
Kara Davis ◽  
Zinaida Good ◽  
Jolanda Sarno ◽  
Astraea Jager ◽  
Nikolay Samusik ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Single Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor

scholarly journals Transcriptome Based Projection of Single Cells to Uncover Development and Heterogeneity of Abnormal Hematopoietic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2520-2520
Author(s):  
Parashar Dhapola ◽  
Mikael Sommarin ◽  
Mohamed Eldeeb ◽  
Amol Ugale ◽  
David Bryder ◽  
...  
Keyword(s):  
Single Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Cell Identity ◽  
Large Patient ◽  
Low Dimensional

Single-cell transcriptomics (scRNA-Seq) has accelerated the investigation of hematopoietic differentiation. Based on scRNA-Seq data, more refined models of lineage determination in stem- and progenitor cells are now available. Despite such advances, characterizing leukemic cells using single-cell approaches remains challenging. The conventional strategies of scRNA-Seq analysis map all cells on the same low dimensional space using approaches like tSNE and UMAP. However, when used for comparing normal and leukemic cells, such methods are often inadequate as the transcriptome of the leukemic cells has systematically diverged, resulting in irrelevant separation of leukemic subpopulations from their healthy counterpart. Here, we have developed a new computational approach bundled into a tool called Nabo (nabo.readthedocs.io) that has the capacity to directly compare cells that are otherwise unalignable. First, Nabo creates a shared nearest neighbor graph of the reference population, and the heterogeneity of this population is subsequently defined by performing clustering on the graph and calculating a low dimensional representation using t-SNE or UMAP. Nabo then calculates the similarity of incoming cells from a target population to each cell in the reference graph using a modified Canberra metric. The reference cells with higher similarity to the target cells obtain higher mapping scores. The built-in classifier is used to assign each target cell a reference cluster identity. We tested Nabo's accuracy on control datasets and found that Nabo's performance in terms of accuracy and robustness of projection is comparable to state-of-art methods. Moreover, Nabo is a generalized domain adaptation algorithm and hence can perform classification of target cells that are arbitrarily dissimilar to reference cells. Nabo could identify the cell-identity of sorted CD19+ B cells, CD14+ monocytes and CD56+ by projecting these unlabeled cells onto labelled peripheral blood mononuclear cells with an average specificity higher than 0.98. The general applicability of Nabo was demonstrated by successfully integrating pancreatic cells, sequenced in three different studies using different sequencing chemistries with comparable or better accuracy than existing methods. Also, it was conclusively demonstrated that Nabo can predict the identity of human HSPC subpopulations to the same accuracy as can be achieved by established cell-surface markers. Having Nabo at hand, we aimed to uncover the heterogeneity of hematopoietic cells from different stages of AML. Nabo showed that AML cells lacked the heterogeneity of normal CD34+ cells and were devoid of cells with HSC gene signature. A large patient-to-patient variability was found where leukemic cells mapped to distinct stages of myeloid progenitors. To ask whether this variability could reflect differences in leukemia-initiating cell identity, we induced leukemia in murine granulocyte-monocyte-lymphoid progenitors (GMLPs) using an inducible model for MLL-ENL-driven AML. On projection, more than 70% of MLL-ENL-activated cells mapped to a distinct Flt3+ subpopulation present within healthy GMLPs. Statistical validity of this projection was verified using two novel null models for testing cell projections: 1) ablated node model, wherein the mapping strength of target cells are evaluated after removal of high mapping score source nodes, and 2) high entropy features model, which rules out the background noise effect. By separating Flt3+ and Flt3- cells prior to activation of the fusion gene and performing in vitro replating assays, we could demonstrate that Flt3+ GMLPs contained 3-4 fold more leukemia-initiating cells (1/1.34 cells) than Flt3- GMLPs (1/4.89 cells), indicating that leukemia-initiating cells within GMLPs express Flt3. Taken together, Nabo represents a robust cell projection strategy for relevant analysis of scRNA-Seq data that permits an interpretable inference of cross-population relationships. Nabo is designed to compare disparate cellular populations by using the heterogeneity of one population as a point of reference allowing for cell-type specification even following perturbations that have resulted in large molecular changes to the cells of interest. As such, Nabo has critical implementation for delineation of leukemia heterogeneity and identification of leukemia-initiating cell population. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document