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We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversionmutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.

Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.

Despite impressive response to front-line treatment, relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL) remains one of the most common causes of death in children with cancer. In pediatric and adult r/r B-ALL, CD19 CAR T cell (CART19) therapy has shown impressive results with over 80% of patients achieving complete remission. However, as an unintended consequence of long-term persistence of CAR T cells and the resulting sustained pressure against CD19 expressing cells, 30 - 70% of initial responders will eventually relapse with CD19 loss. Early identification of patients that will respond successfully to CART19 therapy vs. those at risk for CD19 Neg relapse would support clinical decision making and improve patient care. To address this problem, we profiled by mass cytometry (CyTOF) 25 pre-CART19 samples including patients that achieved a durable complete remission (CR, n = 6), patients that underwent CD19 Neg (n = 11) or CD19 Pos relapse (n = 4) and patients that were refractory to CART19 (n = 4). For 14 of those patients, we also analyzed paired post-CART19 samples: 8 from CD19 Neg relapse, 2 from CD19 Pos relapse, and 4 from refractory tumors. We observed a significant increase in the proportion of leukemic early progenitor non-B cells (called early-non-BI) after CD19 Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. In addition, in the pre-CART19 samples we identified a subpopulation of CD19 Pos pro-B cells that resemble early-non-BI cells. We hypothesized that these pro-B "discordant" cells are able to lose CD19 expression and escape the immune pressure exerted by the CART19, leading to CD19 Neg relapse. Closer analysis revealed lower levels of IKAROS in pro-B discordant cells from patients that underwent CD19 Neg relapse, than in those that achieved a durable CR. To further explore the difference between pro-B discordant cells from patients that achieved a durable CR or underwent CD19 Neg relapse, we performed single cell REAP-seq (RNA expression and protein sequencing) from 3 pre-CART19 CR and 3 pre-CART19 CD19 Neg relapse r/r B-ALL samples. From this analysis, we confirmed a subpopulation of leukemic pro-B like cells with decreased expression of IKZF1 in those patients that underwent CD19 Neg relapse. IKAROS is a transcription factor that plays a crucial role in B cell lineage specification and commitment and is a known B-ALL tumor suppressor. Genetic alterations affecting the IKAROS gene, IKZF1, have been associated with poor response to front-line treatment and they are a factor in relapse risk stratification. However, a role for IKAROS in CART19 failure has not been described. To assess if there is a correlation between the levels of IKAROS and CD19 surface expression, we manipulated IKAROS levels in B-ALL cell lines through RNA silencing and targeted protein degradation approaches. Consistently, we observed that CD19 surface expression diminishes upon IKAROS downregulation. To determine how IKAROS regulates CD19 expression, we performed CyTOF, RNA-seq and ATAC-seq analysis of isogenic B-ALL cell lines with wild-type or reduced (IKAROS KD) IKAROS levels. While the chromatin accessibility to CD19 regulatory regions did not change, CD19 mRNA levels were lower in IKAROS KD cells. Interestingly, we found that IKAROS regulates several genes associated with mRNA splicing. In particular, CD19 mRNA preferentially retains intron 10 in IKAROS KD cells. The inclusion of intron 10 introduces several premature stop codons. Moreover, IKAROS KD B-ALL cells more closely resemble myeloid cells based on their gene expression profiles, suggesting a loss in their B cell identity. In summary, this is the first description of a leukemia-specific subpopulation of CD19 Pos pro-B discordant cells in patients prior CART19 administration. Furthermore, lower levels of IKAROS in this subpopulation correlates with poor response to CART19 therapy. Preliminary molecular analysis suggests that low IKAROS result in retention of CD19 intron 10 and loss of B-cell identity (Figure 1). Future research will focus on in-depth characterization of the myeloid-like phenotype induced by low levels of IKAROS expression, and the impact that intron 10 retention has on CD19 mRNA stability and surface expression. Figure 1 Figure 1. Disclosures Mullighan: Pfizer: Research Funding; Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Barrett: Tmunity Therapeutics: Current Employment. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Mass cytometry is now a well-established method that enables the measurement of 40-50 markers (generally proteins but transcripts are also possible) in single cells. Analytes are detected via antibodies tagged with heavy metal and detected by using a time-of-flight mass spectrometer. Over the past decade, mass cytometry has proven to be a valuable method for immunophenotyping hematopoietic cells with remarkable precision in both healthy and malignant scenarios. This chapter explains in detail how to profile hematopoietic cells by using this high-dimensional multiplexed approach.

Mass cytometry is now a well-established method that enables the measurement of 40-50 markers (generally proteins but transcripts are also possible) in single cells. Analytes are detected via antibodies tagged with heavy metal and detected by using a time-of-flight mass spectrometer. Over the past decade, mass cytometry has proven to be a valuable method for immunophenotyping hematopoietic cells with remarkable precision in both healthy and malignant scenarios. This chapter explains in detail how to profile hematopoietic cells by using this high-dimensional multiplexed approach.

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately, >70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers., Maeve O’Huallachain et al. report a method that enables simultaneous, ultra-high throughput single-cell barcoding for targeted single-cell protein and RNA analysis. They show the utility of their method in analyses of mRNA and protein expression in human and mouse cells.

Insight into the cancer cell populations that are responsible for relapsed disease is needed to improve outcomes. Here we report a single-cell-based study of B cell precursor acute lymphoblastic leukemia at diagnosis that reveals hidden developmentally dependent cell signaling states that are uniquely associated with relapse. By using mass cytometry we simultaneously quantified 35 proteins involved in B cell development in 60 primary diagnostic samples. Each leukemia cell was then matched to its nearest healthy B cell population by a developmental classifier that operated at the single-cell level. Machine learning identified six features of expanded leukemic populations that were sufficient to predict patient relapse at diagnosis. These features implicated the pro-BII subpopulation of B cells with activated mTOR signaling, and the pre-BI subpopulation of B cells with activated and unresponsive pre-B cell receptor signaling, to be associated with relapse. This model, termed 'developmentally dependent predictor of relapse' (DDPR), significantly improves currently established risk stratification methods. DDPR features exist at diagnosis and persist at relapse. By leveraging a data-driven approach, we demonstrate the predictive value of single-cell 'omics' for patient stratification in a translational setting and provide a framework for its application to human cancer.

Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

Glucocorticoids (GCs) remain a backbone component of therapeutic regimens for childhood B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is a strong prognostic marker of relapse making its understanding an important challenge to be addressed to improve overall patients outcome. Healthy B-cell development is characterized by checkpoints where critical regulatory signaling influences the fate of developing B-cells. These stages are vulnerable for leukemic transformation and as we previously demonstrated, the developmental and functional state of B-ALL cells are of critical importance in treatment failure. Using this developmental framework, we examined the effect of GCs on healthy and malignant B-cells to better understand mediators of GC resistance and ways to overcome it. To define the dynamics of the transcriptional networks surrounding B-cell developmental checkpoints, we performed transcriptomic analysis of sorted pre-proB, pro-B and pre-B cells from 3 healthy donors. We found a coordinated upregulation of B cell receptor (BCR) and Glucocorticoid Receptor (GCR) pathways in healthy B cells during their development. Single cell proteomic analysis of these healthy populations confirmed the coordinated expression of GCR with early B-cell phenotypic markers. Furthermore, in vitro treatment of healthy B-cells with the GC dexamethasone (dex) demonstrated cycling pro-B/pre-B cells to be the most sensitive to GC-induced cell death. Given the importance of GCs in B-ALL treatment, we investigated GCs effects and resistance in NALM6 and REH cell lines. NALM6, expressing high levels of GCR, were significantly sensitive to dex treatment in terms of cell cycle arrest and cell death. By contrast, REH cells were resistant to dex treatment as they lack the GCR. Retroviral transfection of NR3C1 (GCR gene) in REH cells (REH GCR) resulted in acquired sensitivity to dex. To explore the potential crosstalk between GCR and BCR pathways, we also treated cells with BCR signaling inhibitor, dasatinib (das), alone or in combination with dex. While both cell lines survived to treatment with das, the combined treatment increased apoptosis compared to dex alone in NALM6 cells (p=0.0179) and REH GCR cells (p=ns). Whole transcriptome sequencing of dex-resistant cells revealed upregulation of BCR downstream signaling as one of the main pathways associated with resistance. The cell line data implicated active BCR signaling as a path to GCs resistance, so we next analyzed 19 B-ALL primary samples by mass cytometry, after in vitro exposure for 48 hrs to same treatments. Across the entire cohort, dex induced a significant reduction in viability (p=0.0007), compared to vehicle. Treatment with das and dex+das also decreased cell viability compared to vehicle (p=0.0038 and p=0.0002) although was not significant to dex alone. Interestingly dex-resistant cells showed a phenotypic modulation compatible with a late pre-B phenotype with increased CD45 and CD20 expression. In addition, surviving cells showed an activation of downstream targets of BCR signaling such as pSYK, pRPS6 and pCREB that was partially blunted by dasatinib treatment. To understand whether the phenotype and signaling modulation induced by dexamethasone also occurs in patients after treatment with GCs, we analyzed minimal residual disease (MRD) cells following 8 days of treatment with GCs from 9 B-ALL patients. This analysis confirmed our previous findings with MRD cells having same late pre-B cells phenotype and signaling profile as in vitro treated cells. Finally, we tested whether targeting of pre-BCR signaling via dasatinib, could overcome resistance in vivo. We evaluated engraftment of luciferase-expressing NALM6 cells at different timepoints after tail vein injection in mice treated with vehicle, dex, das or dex + das. Bioluminescence analysis revealed a significant reduction of early and late engraftment in the dex + das group compared to vehicle-treated mice. Furthermore, mice receiving the combined treatment also experienced a significant survival advantage as assessed by log rank test (p=0.0002). Taken together these data suggest a coordinated interplay between BCR and GCR pathways in healthy and leukemic B cells. GCs-resistant leukemic cells showed a mature pre-B phenotype that is vulnerable to BCR signaling inhibition in vitro and in vivo suggesting new therapeutic options to overcome GC resistance in childhood B-ALL. Disclosures Biondi: Bluebird: Other: Advisory Board; Novartis: Honoraria; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria. Bava: 10x Genomics: Current Employment. Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria.

Single-cell omics provide insight into cellular heterogeneity and function. Recent technological advances have accelerated single-cell analyses, but workflows remain expensive and complex. We present a method enabling simultaneous, ultra-high throughput single-cell barcoding of millions of cells for targeted analysis of proteins and RNAs. Quantum barcoding (QBC) avoids isolation of single cells by building cell-specific oligo barcodes dynamically within each cell. With minimal instrumentation (four 96-well plates and a multichannel pipette), cell-specific codes are added to each tagged molecule within cells through sequential rounds of classical split-pool synthesis. Here we show the utility of this technology in mouse and human model systems for as many as 50 antibodies to targeted proteins and, separately,70 targeted RNA regions. We demonstrate that this method can be applied to multi-modal protein and RNA analyses. It can be scaled by expansion of the split-pool process and effectively renders sequencing instruments as versatile multi-parameter flow cytometers.

The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics.

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force directed layout visualization, defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and aging.

CD19-directed chimeric antigen receptor T cell (CAR-T) therapy has shown impressive results in children and adults with relapsed or refractory B-ALL or diffuse large B-cell lymphoma. However, 30 - 70% of initial responders will eventually relapse with CD19 antigen loss (CD19Neg) (Maude SL, et al. N Engl J Med. 2018). To avoid CD19Neg relapse, patients may undergo a hematopoietic stem cell transplant (HSCT). HSCT is an expensive and often morbid procedure that many physicians would prefer to avoid. The development of tools to accurately predict which patients are at risk for CD19Neg relapse would guide treatment decisions regarding HSCT or alternative therapies. Since CD19Neg relapses also occur in patients treated with other CD19-directed immunotherapies, like blinatumomab (Mejstríková E, et al. Blood Cancer J. 2017), a predictive model to detect patients at risk of CD19Neg relapse would have broader therapeutic impact. To address this problem, we performed CyTOF and RNA-seq analysis from paired patient samples collected before CD19-directed CAR-T administration and after CD19Neg relapse. High dimensional phenotyping by CyTOF clustered patient samples based on their mechanism of CD19 expression loss (frameshift mutation versus expression of intracellular isoforms), even before CAR-T administration. In addition, we identified identical immunoglobulin heavy and light chain RNA sequences before CAR-T administration and after CD19Neg relapse, suggesting that the clones destined to cause relapse are present at the time of CAR-T administration. Altogether, these results support our hypothesis that resistant tumor cells are present before CAR-T administration and could be discovered and interrogated for CD19Neg relapse prediction. To identify cell subpopulations responsible for driving CD19Neg relapse, we used the B cell developmental classifier previously developed in our lab (Good Z, et al. Nat Med. 2018). We observed a significant increase in the Early-non-BI population (CD38Pos CD24Pos CD19Neg CD20Neg CD3Neg CD16Neg CD61Neg cells) after CD19Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. Since our classifier relies on CD19 to classify cells, we compared the resulting classification of cells when CD19 was included or excluded in the classifier. This change had minimal impact in cell classification from healthy bone marrow controls. However, when applied to the samples collected before CAR-T administration, we found a subpopulation of CD19Pos Pro-B cells that classified as Early-non-BI cells when CD19 was excluded from the classification. We hypothesize that these Pro-B "discordant" cells are those that lose CD19 expression to escape the immune pressure exerted by the CD19-directed CAR-T and mediate CD19Neg relapse. Further, we found Pro-B "discordant" cells in 77% of independent cohort of 22 B-ALL samples collected at the time of diagnosis, suggesting these cells exist in de novo B-ALL. We likewise identified a CD19Neg IgMPos Early-non-BI subpopulation in 4 healthy bone marrow and further studies are ongoing to characterize these cells. We continue to interrogate this candidate population as that responsible for CD19Neg relapse after CAR-T cell therapy. In addition, we performed differential expression analysis between paired samples collected before (CD19Pos) and after (CD19Neg) CAR-T therapy. Through the application of the developmental classifier, we identified that CD19 loss is associated with upregulation of key B cell transcription factors IKAROS, PAX5 and glucocorticoid receptor in the pre-pro-B to Pre-B stages. Moreover, after CD19 loss, there are also increases in levels of phosphorylated proteins pSYK, pSRC and pSTAT5, involved in IL7 receptor and pre-BCR signaling pathways, essential for B cell development. This suggests that CD19Neg cells activate unique tumorigenic pathways that may provide novel therapeutic opportunities. Exploration and validation of these therapeutic targets could significantly improve clinical outcome and care of patients with CD19Neg B-ALL. In conclusion, these results support the feasibility to predict patients at risk for CD19Neg relapse together with the mechanism behind it. Future studies will be conducted to confirm unique tumorigenic pathways in CD19Neg B cells and determine their therapeutic potential. Disclosures Mullighan: Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Amgen: Honoraria, Other: speaker, sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Loxo Oncology: Research Funding. Grupp:Humanigen: Consultancy; CBMG: Consultancy; Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy.

Glucocorticoids (GCs) have a crucial role in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is considered a strong prognostic marker of relapse, which occurs in about 20% of pediatric B-ALL patients. GCs exert therapeutic effects by inducing cell cycle arrest and eventually, cell death. However, their precise mechanisms of action and resistance are not fully understood. We have previously demonstrated the importance of developmental state in treatment failure in B-ALL (Good Z Nat Med 2018 and Bendall SC Cell 2014), specifically at the developmental transition between pro-B to pre-B cells. Using this developmental framework, we examined the effect of GCs on healthy and malignant early B cells progenitors to better understand mediators of GC resistance. To understand the transcriptional programs mediating early B-cell differentiation, we sorted three B-cell developmental populations (pre-pro-B, pro-B/pre-B I and pre-B II) and performed RNA-seq analysis. Ingenuity pathway analysis revealed a coordinated upregulation of B cell developmental genes and Glucocorticoid Receptor (GCR) pathway genes in healthy pre-B I cells, suggesting a role of GCs in healthy B-cell development. We confirmed expression of the GCR in healthy pre-B I cells by mass cytometry (CyTOF). Further, in vitro treatment of healthy B-cells with GCs (dexamethasone, dex 1 uM) demonstrated pre-B I cycling cells to be most sensitive to GC-induced cell death. Given the importance of GCs in B-ALL treatment, we investigated the GCR pathway and its relationship with B-cell development in 18 B-ALL primary samples (14 diagnosis, 4 relapse; including Ph+=1; MLL rearranged=2; TEL-AML translocated=3). Samples were profiled by CyTOF with a 40-antibody panel including surface markers, signaling molecules, transcription factors, apoptosis and cell cycle molecules. To better comprehend the relation of GCR pathway with BCR signaling, primary cells were analyzed after 48 hours of treatment with dex (1uM), SRC/ABL inhibitor dasatinib (das, 100 nM) or their combination. Similar to results obtained in healthy B-cells, we found the highest expression of GCR in the pre B-I cells in all patients (p Interestingly, we also observed consistent patterns of phenotypic modulation in dex-resistant cells and to understand how these were related to B-cell development we performed developmental classification (Good Z Nat Med 2018) of leukemic cells in dex-treated versus untreated conditions. This analysis revealed a large decrease of early B-cell progenitors and corresponding increase of more mature subsets of either B and non-B cells (false discovery rate, FDR To determine if this effect was directly related to GCR expression, we compared results between a GCR+ cell line (NALM6) and GCR- cell line (REH). Consistent with the primary samples, the combination of dex with das more effectively killed NALM6 cells yet the REH were unresponsive to either treatment. However, overexpressing GCR in REH cells reversed dex-resistance, also becoming more sensitive to the combined treatment with das. Similar to primary cells, NALM6 and REH GCR+ cells showed a phenotypic change when treated with dex, that will be further investigated to clarify the relationship between the dex-induced phenotype and its apoptotic effect. In summary, these data suggest a dual role of glucocorticoids on early B-cells; mediating both apoptosis and differentiation in relationship to cell cycle status and GCR level. These findings may carry therapeutic implications and suggest cell types vulnerable to BCR signaling inhibition as a therapeutic intervention to overcome GCs resistance. Disclosures Nolan: Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold.

Numerous studies have demonstrated the association between minimal residual disease (MRD) positivity and risk of eventual relapse for children with B cell precursor acute lymphoblastic leukemia (BCP ALL). Yet despite varied methods to detect MRD (flow cytometry, PCR, sequencing), we still lack knowledge of the biology of drug resistant cells that mediate relapse. Performing single cell proteomic analysis using mass cytometry in a clinically annotated cohort of diagnostic samples, we have identified a phosphorylated ribosomal protein S6 (prpS6) signaling phenotype associated with MRD positivity and relapse. This phenotype has been observed in matched diagnostic and MRD samples and notably, in matched diagnosis/relapse pairs, we have observed an enrichment for this population at relapse. Methods Diagnostic BCP ALL samples from patients treated on the AIEOP protocols or healthy bone marrow were assayed either in basal condition or after ex vivo treatment with tyrosine kinase inhibitors (Dasatinib, BEZ-235). Samples were analyzed using mass cytometry for expression of 40 functional and/or phenotyping proteins. Mass cytometry data was processed and analyzed using Cytobank. Statistical analysis was performed using GraphPad Prism software. Significance was determined using student's t-test or one way ANOVA. Results Of the nine functional proteins measured in this study, rpS6 demonstrated, in resting cells from patients, the most variable levels of phosphorylation. Across the cohort the frequency of cells with activated rpS6 in the basal state varied between 4%-86% (median 24.7%), depending on the patient. This level was significantly higher than in healthy bone marrow controls (median 10.7%; p=0.0009). In BCP ALL samples the cells with activated rpS6 expressed higher levels of CD19 (p=0.0001), IgH (p=0.0059), and PAX5 (p=0.0021) when compared to cells without activated rpS6, suggesting a more mature phenotype for the prpS6+ blasts. These cells also had significant activation of pCREB (p Patients with intermediate or high risk MRD status (per clinical protocol definitions) had an increased frequency of cells with activated rpS6 at diagnosis compared with patients with standard risk MRD (30% vs 13.9%; p=0.02). For patients for whom long term follow up data was available (n=31), patients that went on to relapse (n=9) compared to those in remission (n=22) had a statistically significant increased frequency of cells with activated prpS6 (p=0.0391). To determine whether activation of prpS6 was important for cell survival, we identified two cells lines amongst 7 BCP ALL cell lines that contained this high basal prpS6 subpopulation: NALM6 and SUP-B15. Treatment of these cells with Dasatinib in combination with the BEZ-235 or the MEKi PD0325901 resulted in decreased viability (6%-25% in TKI treated vs. 84% in untreated) and decreased activation of prpS6 and pCREB at 72 hours. These results support the hypothesis that the prpS6 high phenotype is important for cell survival. Given the association of activated rpS6 at diagnosis with detectable MRD and relapse, we examined an additional cohort of 9 matched samples from diagnosis Day 8 and Day 15 post induction therapy. In these samples, it was noted that by Day 8, there was a significant enrichment for cells with activated rpS6 (p=0.0009). In paired diagnosis-relapse samples, in 5 of 6 pairs, there was an enrichment for cells with activated rpS6atrelapse, all suggesting that rpS6 is associated with cells that are drug resistant. Mean prpS6 was significantly higher at relapse compared to diagnosis (p=0.035). Further studies will be critical to determine whether the rpS6 signature is proximal to causal events driving resistance. Significance We describe biochemical features of a cellular population observed at diagnosis across genetic subtypesassociated with MRD positivity and relapse that is enriched in early MRD and relapse samples in BCP ALL. Ribosomal protein S6 is a target of PI3K, MEK/ERK signaling and the B cell receptor pathway. Inhibition of this signaling results in cell death in model cell lines and suggests a role for therapy directed at this target in BCP ALL. Disclosures Davis: Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

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.

Background: Flow cytometric immuno-phenotyping of lineage-associated antigens is used in the diagnosis of BCP ALL to distinguish neoplastic B-cells. However, the resultant immunophenotypic expression patterns are inadequate to inform prognosis or choice of therapy. Mass cytometry allows for multi-parametric analysis of single cells to distinguish phenotypic and functional features of subpopulations from leukemia samples. Mass cytometric analysis of pediatric Ph+ BCP ALL constructs a novel model of ALL organized along the normal B cell developmental trajectory (Davis et al., Cell 2014). Leukemic cells share phenotypic features with their normal early B cell counterparts. Critical survival and proliferation signaling is also linked to phenotypic state. Further, the developmental state of leukemic populations common among the patients impacts in vitroresponse to inhibition of BCR-ABL kinase signaling. Methods: Mononuclear cells from diagnostic bone marrow samples were obtained from pediatric patients with Philadelphia chromosome positive BCP-ALL under informed consent (n=21) or healthy controls (n=5). Mass cytometry analysis of 40 proteins was performed at baseline state and perturbed state (IL-7, TSLP, anti-m, dasatinib, tofacitinib, BEZ-235) as previously described (Bendall et al., Science 2011). Analysis was restricted to progenitor and blast populations. Healthy bone marrow samples were gated as previously described along the trajectory of developing B cells (Davis et al., Cell2014). These populations were used as the foundation for a classifier in which each leukemia cell was assigned to its nearest healthy population based on a distance metric (Mahalanobis in nine dimensions). Results: Compared to healthy bone marrow controls, and as expected, ALL samples displayed overexpression of early B cell immunophenotypic markers including CD10 (healthy mean counts 3.83 vs. leukemic 283.3, p=0.03), CD34 (6.26 vs. 80.7; p=0.03), and TdT (2.03 vs.18.9; p=0.002). Leukemic cells expressed lower levels of CD45 and IgM compared to healthy developing B cells. Extended phenotyping revealed conserved patterns of protein expression consistent with different developmental stages in B cell development. We have previously identified the precise developmental ordering of human B cell fractions based on the combined expression of CD34, CD38, CD24, TdT (Davis et al., Cell 2014). ALL samples showed increased numbers of cells occupying B cell progenitor compartments compared to healthy bone marrow controls. To formalize this observation, a single-cell classifier was constructed based on the developmental trajectory of healthy B cells. Each leukemic cell was assigned its most related healthy B cell population based on the expression of nine developmental proteins. Across all samples, the size of the pre-proB (CD34+CD38+TdT+) and proB (CD34+CD38+TdT+CD24+) compartments expanded (12% and 33% in ALL vs. 1% and 2% in healthy, respectively) at the expense of progenitor and preB cell compartments. Interestingly, within a given sample, cells may expand within more than one progenitor compartment such that each leukemia had a corrupted, but distinct B cell developmental trajectory. In two diagnosis-relapse pairs, the relapse sample occupied a more mature phenotype compared to its diagnostic partner. Within the developmental compartments, blast cells retained functional features of their healthy counterparts. Blasts within the ProB cell compartment displayed higher basal levels of pSTAT5, pS6, p4EBP1 and pCreb than blasts in other developmental compartments. These cells were more proliferative based on higher mean expression of Ki67. In healthy bone marrow, cells in this developmental state are characterized by ligand-independent STAT5 activation. Indeed the leukemia’s overall level of pSTAT5 correlated with the percentage of cells in the ProB state (R2=0.71). Similarly, in the leukemic samples, patients with a high percentage of cells in this state were less able to respond to inhibition of STAT5 with the tyrosine kinase inhibitor, Dasatinib than cells in other developmental compartments. Conclusions: Deep proteomic profiling of BCP ALL establishes a single-cell classification linking phenotype with functional attributes of leukemic cells. This data demonstrates that leukemic cells are more or less sensitive to therapeutic intervention based on their developmental state. Disclosures Bendall: Fluidigm: Consultancy. Simonds:Fluidigm: Consultancy, Equity Ownership. Nolan:Fluidigm, Inc: Consultancy, Equity Ownership.

Acute myeloid leukemia (AML) can present with multiple concurrent subclones at diagnosis. Subclone-specific mutations may confer resistance to molecular-targeted drugs through loss of antigen expression or rewiring of intracellular signaling pathways, leading to relapse. Deep sequencing approaches improve the detection of rare subclones, but the ability to prospectively identify subclones (i.e. without relapse material) is limited, and the effects of subclone-specific mutations on phenotype are poorly understood. In the course of a broader study of oligoclonal pediatric AML patients, we focused investigation on a diagnosis bone marrow sample from a patient harboring 3 presumed subclones (two with distinct NRAS-G12D, NRAS-G13D mutations) as determined by whole-genome sequencing (WGS). We employed a combination of 31-parameter mass cytometry, deep sequencing, FACS sorting, and computational modeling to produce a detailed profile of the subclonal genotypes and phenotypes in this patient. The 3 anticipated subclones did not correlate with a clear subset of surface markers in the mass cytometry analysis. Instead, we observed a single continuous ‘differentiation trajectory’ from progenitor-like to monocyte-like blasts. To dissect this trajectory, 7 distinct myeloid/progenitor subsets were FACS-sorted, plus T/B cells as a non-leukemic control. For each subset we performed capture-based deep sequencing of 307 tumor-specific variants (Tier 1: 13; Tier 2: 33; Tier 3: 261; median depth: 1420x). Shifts in allele frequencies among the FACS-sorted subsets provided critical information to a Bayesian mixture modeling algorithm, allowing identification of 5 subclones, as opposed to the 3 subclones anticipated by WGS. The inferred subclonal genotypes were validated and further refined by targeted single-cell Sanger sequencing of multiple Tier 3 loci. Although all 5 subclones were present throughout the differentiation trajectory, some were enriched in certain phenotypic states or ‘reservoirs’. For example, the NRAS-G12D subclone was enriched in a progenitor-like subset, but its daughter subclone, which harbored an additional mutation in RAC2 (implicated in HSC engraftment), was enriched in the more differentiated subsets, suggesting an opposing effect. Notably, the T/B cell population harbored 2 tumor-specific Tier 1 mutations at >15% allele frequency, suggesting its presence in a preleukemic, multilineage-competent HSC. Taken together, subclone-specific mutations appear to skew cells toward either progenitor or mature phenotypes, but the developmental trajectory enforced by parental mutations is resistant to change. Furthermore, characterization of subclones is possible at diagnosis, and may improve the selection of targeted therapies. *Contributed equally: EFS, SCB & ALG Citation Format: Erin F. Simonds, Sean C. Bendall, Amanda L. Gedman, Jacob H. Levine, Kara L. Davis, Harris G. Fienberg, Astraea Jager, El-ad D. Amir, Ina Radtke, Wendy J. Fantl, Dana Pe'er, James R. Downing, Garry P. Nolan. Deep sequencing of immunophenotypically distinct subsets in acute myeloid leukemia reveals reservoirs of genetically distinct subclones along a conserved differentiation trajectory. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2013-3178

Increasing evidence suggests tumors are maintained by cancer stem cells, however their nature remains controversial. In a HoxA9-Meis1 (H9M) driven model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy–stem/progenitor cells (Lin−kit+), and committed progenitors of the myeloid (Gr1+kit+) and lymphoid lineages (Lym+kit+). Each compartment clonally recapitulated the original range of tumor cell immunophenotypes in vivo, including cells with a less-differentiated immunophenotype. These distinct populations largely shared signaling networks, and in vivo pharmacologic targeting of shared pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, these data show that H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, in some malignancies, tumor-initiation ability (or “cancer-stemness”) can represent a targetable, cellular state that can exithat exists independently of distinct immunophenotypic definition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3307. doi:1538-7445.AM2012-3307

Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, this suggests that in certain malignancies tumor-initiation activity (or "cancer stemness") can represent a cellular state that exists independently of distinct immunophenotypic definition.