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

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.

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LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2348.2348 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2348-2348

Author(s):

Hirotaka Kawano ◽

Tomotoshi Marumoto ◽

Michiyo Okada ◽

Tomoko Inoue ◽

Takenobu Nii ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Lymphoblastic Leukemia ◽

Embryonic Stem ◽

Common Marmoset ◽

Hematopoietic Stem ◽

Helix Loop Helix ◽

Cd34 Cells

Abstract Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

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Analysis of Single Cell Transcriptomics in Paired Pediatric T-ALL Samples Collected at Diagnosis and Following End of Induction Therapy Reveals an MRD-Associated Stem Cell Signature

Blood ◽

10.1182/blood-2021-153841 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1311-1311

Author(s):

Swati S Bhasin ◽

Beena E Thomas ◽

Ryan J Summers ◽

Debasree Sarkar ◽

Hope L Mumme ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

B Cell ◽

Single Cell ◽

Communication Networks ◽

Residual Disease ◽

Differential Expression Analysis ◽

Gene Signature ◽

Patient Specific ◽

Immune Microenvironment

Abstract Introduction Despite recent improvement in outcomes for de novo disease, pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains challenging to treat at relapse. Investigation into genomic markers of treatment response and therapy resistance offers an opportunity to further enhance outcomes for these patients. We previously identified a T-ALL blast-associated gene signature at diagnosis (Dx) and characterized the immune microenvironment in Dx T-ALL marrow samples using single cell transcriptome analysis (Bhasin et al. Blood 2020(ASH)). This approach allowed us to generate a granular expression map of both the T-ALL landscape and the Dx bone marrow (BM) immune microenvironment. Here we expand this work by evaluating samples collected from the same patients Dx and End of Induction (EOI) BM samples from pediatric T-ALL patients. The use of paired samples provides insight into treatment-induced changes in the microenvironment. Further, the inclusion of both minimal residual disease (MRD) positive and MRD negative samples allowed us to compare differences between these groups. Methods Using the 10X genomics platform, we profiled the single cell transcriptome of ~18,000 BM and immune microenvironment cells from viably frozen samples collected from T-ALL patients at Dx or EOI. Five paired Dx and EOI samples and one EOI sample from a patient with relapsed T-ALL were evaluated, for a total of 11 samples. Three paired samples were MRD positive at EOI and two were MRD negative; the relapsed sample was MRD negative. Cell clustering was performed using the Seurat package and differential expression analysis was performed using R/Bioconductor packages (Hao et al. Cell 2021). Cell communication analysis was conducted using the CellChat R tool (v 1.0.0) to infer cell-cell communication within the EOI MRD positive and MRD negative subsets and compare their communication networks (Jin et al. Nature Comm 2021). Results Using our previously described blast-associated gene signature (Bhasin et al. ASH 2020) we were able to identify residual blast populations at EOI in MRD-positive samples. Comparative analysis of gene profiles at Dx and EOI showed significant changes in the microenvironment cell populations with highest increase in erythroid cell populations after induction therapy. The gene expression profiles were significantly different for immune cells at Dx and EOI and the relapsed sample had greater similarity to the Dx samples indicating a persistent immunosuppressive environment. Clustering analysis of the EOI samples (3 MRD positive and 2 MRD negative) demonstrated the presence of patient specific blast cells in MRD positive samples that retained patient-specific transcriptomeheterogeneity at EOI (Fig.1A). Analysis of communication networks between different cell types based on receptor and ligand expression levels between different cell types identified a CD34 + cluster of stem cells that had different interactions with other immune populations in the MRD positive and negative subsets. Differential expression analysis between the MRD positive and MRD negative cells in this CD34 + stem cell cluster identified higher expression of myeloid associated genes such as CEBPB, CEBPD, AZU1 in the MRD negative group relative to the MRD positive cells, which showed higher expression of B-cell related genes such as IGHM, VPREB1, CD79A/ B along with upregulation of P13K signaling in B-lymphocytes, B-cell receptor signaling and autophagy pathways. Analysis of upstream regulators based on the differential gene signature between the MRD positive and MRD negative group demonstrated upregulation of MYC and TCF3 activity and inhibition of TGFB1, CSF3 and CEBPA in MRD positive compared to MRD negative samples (Fig.1B). Conclusions: Leukemic blasts exhibit patient-specific gene expression signatures that are present at EOI in MRD positive samples. Exploration of the impact of minimal residual disease at EOI revealed differential gene expression patterns in stem cells from MRD positive samples, characterized by activation of B cell related signaling pathways and regulators such as MYC and TCF3. In contrast, a more myeloid-like expression signature was observed in stem cells from MRD negative samples. These findings open the avenues for exploration of therapeutic targets of T-ALL progression. Figure 1 Figure 1. Disclosures DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

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Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3759.3759 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3759-3759

Author(s):

Abdulmohsen M Alruwetei ◽

Hernan Carol ◽

Rosemary Sutton ◽

Glenn M Marshall ◽

Richard B Lock

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Clinical Outcome ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Relapse Prediction ◽

Pediatric All ◽

Selection Of

Abstract Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance. Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo. Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v122.21.sci-22.sci-22 ◽

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.

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Dasatinib Shows Therapeutic Potential in the Murine Xenograft Model for TCF3 rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v124.21.3718.3718 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3718-3718 ◽

Cited By ~ 1

Author(s):

Kyle Lenz ◽

Sarah Thompson ◽

Natalya A. Goloviznina ◽

Jianya Huan ◽

Dorian H LaTocha ◽

...

Keyword(s):

Clinical Trials ◽

B Cell ◽

Therapeutic Potential ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Patient Costs ◽

Nsg Mice ◽

Murine Xenograft Model ◽

Ic50 Values

Abstract Transcription Factor 3 (TCF3) rearrangements are a recurring chromosomal abnormality in B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) occurring in approximately five percent of pediatric ALL. Historically, the majority of these patients carried a poor prognosis, but advances with more intensive cytotoxic chemotherapy have improved the survival rate while exposing patients to increased short and long-term toxicities. Two genetic rearrangements produce the chimeric transcription factors, TCF3-PBX1 t(1;19)(q23;p13) and a much rarer TCF3-HLF t(17;19)(q22;p13). Sadly, TCF3-HLF remains an extremely difficult disease to treat with few, if any known survivors. Although it is unknown how these translocations lead directly to disease, it is established that they do result in diseases arrested in a later stage of B-cell differentiation and pre-B cell receptor (pre-BCR) dependence. Recently, we highlighted the concept of targeting the pre-BCR pathway for therapeutic potential using dasatinib (Sprycel). Here, we further examine dasatinib effectiveness in the murine xenograft model for TCF3-rearranged ALL. Methods: Primary patient samples were obtained with written informed consent approved by the Institutional Review Board of Oregon Health and Science University and processed. Mononuclear cells were separated by Ficoll and exposed to increasing concentrations of dasatinib. Inhibitory Concentration of fifty-percent viability (IC50) was calculated for each sample. The median IC50 for over four hundred acute leukemic samples interrogated by this assay was calculated to approximately 100nM. Samples with IC50 values below 30nM were deemed hypersensitive to dasatinib. For xenografts, frozen viable primary patient samples were thawed and grafted via tail-vein into NOD/SCID/IL-2rgnull(NSG) mice 24 hours after sub-lethal irradiation with 200 cGy. Upon engraftment, and in vivo expansion, animals were euthanized and leukemic cells recovered from the spleen were then injected in secondary recipients. One week after injection the mice were divided into two groups and treated by oral gavage with dasatinib at 50mg/kg/dose daily or citrate control for 5 days per week. Treatment continued until the day of sacrifice (4-20 weeks). Peripheral blood engraftment was monitored weekly starting on week 3 by flow cytometry analysis using anti-human CD19 and CD45 (hCD19-APC, hCD45-FITC) versus anti-murine CD45 (mCD45-PerCP-Cy5.5). Flow cytometric data was analyzed using FACS/AriaIII. Results: Screening over one hundred BCP-ALL samples identified that approximately ten percent of these samples show hypersensitivity to dasatinib. TCF3-rearranged ALL and BCR-ABL1 ALL had a majority of samples with IC50's less than 10nM. Throughout all known subsets of ALL except ETV6-RUNX1, there also appeared to be individual samples that have IC50 values less than 30nM, suggesting significant sensitivity to this drug. Of these, three individual TCF3-rearranged ALL samples were identified and xenografted into NSG mice, expanded and injected into secondary recipients. All dasatinib treated cohorts showed significantly less leukemic peripheral blood chimerisms as compared to their vehicle control counterparts. Further, in vitro treatment of xenografted cells with dasatinib indicated inhibition of the pre-BCR by decrease in pan-phospho-SRC. Intriguingly, dasatinib did not completely abolish disease in all TCF3-rearranged ALL, suggesting other important mechanisms for cell viability. Conclusions: These studies show in vivo therapeutic benefits of dasatinib as treatment for TCF3-rearranged ALL, and open the possibility of adding this drug to their treatment. Further studies are underway to address the mechanisms of dasatinib sensitivity of other subsets of ALL identified in our screen in hopes of adding targeted therapies to their treatment. Figure 1 Figure 1. Disclosures Druker: Molecular MD: Consultancy, Equity Ownership, Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. Other; Bristol-Myers Squibb: Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other. Off Label Use: Dasatinib use as potential therapy in ALL.

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Preclinical Evidence for the Efficacy of CD79b Immunotherapy in B Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-148429 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2388-2388

Author(s):

Lennart Lenk ◽

Dorothee Winterberg ◽

Fotini Vogiatzi ◽

Anna Laqua ◽

Lea Spory ◽

...

Keyword(s):

B Cell ◽

Therapeutic Target ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Advisory Board ◽

Research Support ◽

Nsg Mice ◽

Significant Survival ◽

Survival Prolongation

Abstract Patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL) have a favorable prognosis, yet current treatment protocols are based on intensive cytotoxic chemotherapy and therapy options are limited when patients relapse. Novel immunotherapy approaches are therefore needed. We recently reported that the preB cell receptor signaling unit CD79a (known as Igα) is crucial for BCP-ALL engraftment in vivo, particularly in the central nervous system (CNS) employing BCR-ABL + and E2A-PBX1 + patient derived xenograft (PDX) models (Lenk et al., Communications Biology, 2021). CD79a forms a heterodimer with CD79b (known as Igß), which is also expressed on the surface of mature B cells as part of the B cell antigen receptor. Accordingly, the CD79b antibody drug conjugate (ADC) Polatuzumab Vedotin (PolVed) has shown therapeutic efficacy in the treatment of refractory/relapsed (r/r) diffuse large B cell lymphoma. Moreover, CD79a/CD79b may also be present on the cell surface at the pro/pre B cell stage. We therefore hypothesize that CD79b can serve as a therapeutic target in BCP-ALL. First, to substantiate that CD79b is important for BCP-ALL engraftment in vivo, a murine/murine transplantation model was applied. B cell precursors were isolated from mice harboring CD79b with a non-functional signaling domain (CD79b-ITAM-KO) or wildtype mice, malignantly transformed with a BCR-ABL fusion construct and transplanted into NSG-mice. Whereas control cells caused overt leukemia in all animals within 25 days, no animal injected with CD79b-ITAM-KO cells had developed leukemia by the time of sacrifice at 162 days (P<0.001). To investigate the frequency of surface (s)CD79b expression in BCP-ALL patients, we measured sCD79b levels via flow cytometry in diagnostic samples of pediatric BCP-ALL patients with different cytogenetic backgrounds. We detected sCD79b-positivity (defined as ≥10% sCD79b + BCP-ALL cells) in 23/94 patients including BCR-ABL +, E2A-PBX1 +, MLL-rearranged (MLLr), TEL-AML1 + and B-other BCP-ALL patients indicating a population of sCD79b + patients within different cytogenetic BCP-ALL subgroups (Figure 1a). To validate CD79b as a therapeutic target, we applied an unconjugated monoclonal CD79b antibody (CD79b-mAB) to PDX mice bearing either pediatric BCR-ABL + or E2A-PBX1 + PDX samples with high sCD79b expression (49.6% sCD79b + cells and 37.7% sCD79b + cells, respectively). Treatment was initiated one day after BCP-ALL injection, modelling a minimal residual disease (MRD) situation. Therapy with CD79b-mAB (1mg/kg) resulted in a mild reduction of leukemia burden in the spleen (SP) and bone marrow (BM) of PDX mice and a significant reduction of CNS-involvement in both PDX-models as compared to control treated mice (P<0.05 and P<0.01, respectively). To test the hypothesis that treatment of BCP-ALL with a CD79b-ADC outperforms CD79b-mAB, we applied PolVed (1mg/kg) in NSG mice bearing the same E2A-PBX1 + and BCR-ABL + PDX cells. PolVed therapy resulted in a significant reduction of BCP-ALL engraftment in SP, BM and CNS (P<0.01, respectively) and a significant survival prolongation compared to control treated mice in both models (P<0.01, respectively). Of note, 4/5 PolVed-treated E2A-PBX1-PDX animals were free of leukemia by the time of sacrifice (236 days) (Figure 1B). To test the efficacy of PolVed on different BCP-ALL subgroups, we conducted a preclinical phase II-like PDX study, using sCD79b high and sCD79b low PDX samples (defined by sCD79b-expression above or below the median) (5E2A-PBX1 +, 3 BCR-ABL +, 2 MLLr, 1 E2A-HLF + and 1 ETV6-NTRK3 +). Two NSG mice per patient were injected with PDX cells, randomly assigned into treatment groups and PolVed therapy was initiated when 1% PDX-cells were detected in the peripheral blood, modelling an overt leukemia situation. sCD79b low PDX mice did not respond to PolVed treatment (Figure 1c), but we detected a response to therapy and a significant survival prolongation in 5/6 sCD79b high PDX samples irrespective of the cytogenetic background (P<0.05) (Figure 1d). Taken together, our data indicate that a subgroup of BCP-ALL patients is sCD79 + positive and may respond to PolVed treatment. Therefore, we suggest CD79b as a novel therapeutic target in BCP-ALL and propose PolVed as a potential therapeutic agent in r/r disease. *LL and DW contributed equally to this work Figure 1 Figure 1. Disclosures Lenk: OSE Immunotherapeutics: Research Funding. Richter: HTG Molecular Diagnostics, Inc.: Current Employment, Research Funding. Schrappe: SHIRE: Other: research support; JazzPharma: Honoraria, Other: research support; Servier: Honoraria, Other: research support; SigmaTau: Other: research support; Novartis: Honoraria; JazzPharma: Honoraria; Novartis: Honoraria, Other: research support; Servier: Honoraria; Amgen: Other: research support. Cario: Novartis: Other: Lecture Fee. Brüggemann: Incyte: Other: Advisory Board; Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Schewe: Jazz Pharmaceuticals: Other: Advisory Board; SOBI: Other: Advisory Board; Bayer: Other: Advisory Board; OSE Immunotherapeutics: Research Funding.

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Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽

10.1182/blood.v120.21.4295.4295 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4295-4295

Author(s):

Jae-Hung Shieh ◽

Tsann-Long Su ◽

Jason Shieh ◽

Malcolm A.S. Moore

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Culture System ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Cd34 Cells ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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Single cell eQTL analysis identifies cell type-specific genetic control of gene expression in fibroblasts and reprogrammed induced pluripotent stem cells

Genome Biology ◽

10.1186/s13059-021-02293-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Drew Neavin ◽

Quan Nguyen ◽

Maciej S. Daniszewski ◽

Helena H. Liang ◽

Han Sheng Chiu ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Population Genetics ◽

Genetic Variation ◽

Single Cell ◽

Pluripotent Stem Cells ◽

Individual Cell ◽

Cell Type ◽

Cell Type Specific ◽

Induced Pluripotent

Abstract Background The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) has provided a foundation for in vitro human disease modelling, drug development and population genetics studies. Gene expression plays a critical role in complex disease risk and therapeutic response. However, while the genetic background of reprogrammed cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of individual cells which would provide significant resolution. By integrating single cell RNA-sequencing (scRNA-seq) and population genetics, we apply a framework in which to evaluate cell type-specific effects of genetic variation on gene expression. Results Here, we perform scRNA-seq on 64,018 fibroblasts from 79 donors and map expression quantitative trait loci (eQTLs) at the level of individual cell types. We demonstrate that the majority of eQTLs detected in fibroblasts are specific to an individual cell subtype. To address if the allelic effects on gene expression are maintained following cell reprogramming, we generate scRNA-seq data in 19,967 iPSCs from 31 reprogramed donor lines. We again identify highly cell type-specific eQTLs in iPSCs and show that the eQTLs in fibroblasts almost entirely disappear during reprogramming. Conclusions This work provides an atlas of how genetic variation influences gene expression across cell subtypes and provides evidence for patterns of genetic architecture that lead to cell type-specific eQTL effects.

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Mapping single-cell atlases throughout Metazoa unravels cell type evolution

10.1101/2020.09.28.317784 ◽

2020 ◽

Cited By ~ 1

Author(s):

Alexander J. Tarashansky ◽

Jacob M. Musser ◽

Margarita Khariton ◽

Pengyang Li ◽

Detlev Arendt ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Single Cell ◽

Cell Types ◽

Tree Of Life ◽

Transfer Cell ◽

Cell Type ◽

Germ Layers ◽

Animal Evolution ◽

Definition Of

AbstractComparing single-cell transcriptomic atlases from diverse organisms can provide evolutionary definition of cell types, elucidate the origins of cellular diversity, and transfer cell type knowledge between species. Yet, comparison among distant relatives, especially beyond a single phylum, is hindered by complex gene histories, lineage-specific inventions, and cell type evolutionary diversifications. Here, we develop a method to enable mapping cell atlases throughout Metazoa spanning sponge to mouse. Within phyla, we identify homologous cell types, even between distant species, with some even emerging from distinct germ layers. Across phyla, we find ancient cell type families that form densely interconnected groups, including contractile and stem cells, indicating they likely arose early in animal evolution through hierarchical diversifications. These homologous cell types often substitute paralog expressions at surprising prevalence. Our findings advance the understanding of cell type diversity across the tree of life and the evolution of associated gene expression programs.

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In Vitro Culture Reveals Microenvironment-Dependent Growth, Heterogeneity and Hierarchical Structure of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells

Blood ◽

10.1182/blood.v118.21.1483.1483 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1483-1483

Author(s):

Jae Hung Shieh ◽

Peter G Steinherz ◽

Jason Shieh ◽

Malcolm A.S. Moore

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Hierarchical Structure ◽

Culture System ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Cd34 Antigen ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1483 In Vitro culture reveals microenvironment-dependent growth, heterogeneity and hierarchical structure of primary human pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL) cells. J.-H. Shieh1, P. Steinherz2, J Shieh3 and M. A.S. Moore1. 1Moore Laboratory. Cell Biology Program and 2Leukemia and Lymphoma Studies, Department of Pediatrics, Memorial-Sloan Kettering Cancer Center, New York, NY. 3Department of Biology, Brandeis Univ., Waltham, MA Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children. Although this pediatric pre-B ALLs are treatable, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. To address this issue, we systemically evaluated the effects of various tissue culture parameters to the growth of primary pre-B ALL cells. A serum-free MS-5 cells (a murine bone marrow stromal cell line) co-culture system is capable of expanding the pre-B ALL CD34+CD19+ cells and supporting their differentiation to CD34−CD19+ B cells. This expansion requires a contact between the stromal cells and the pre-B ALL cells, and is inhibited by fetal bovine serum and IL-6 in a dose-dependent manner. c-Kit ligand and Flt3 ligand can reverse the IL-6 inhibition. Expansion of individual CD34+CD19+ cells revealed a hierarchical structure with respect to CD34 antigen expression and an heterogeneity in cell proliferation. When the pre-B ALL cells were sorted into CD34dim and CD34bright populations, the CD34dim cells were capable of a faster proliferation but gradually lost their CD34 antigen. In contrast, the CD34bright cells were more slowly proliferating and retained their CD34 antigen. We transduced the B-ALL cells with a fusion gene expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL). These GFP-Lu-pre-B ALL cells display the similar in vitro characteristics and in vivo xenograftment to NOD/SCID IL2R gamma null (NSG) mice as the non-transduced pre-B ALL cells. One hundred, 103, 104 or 105 GFP-LU-pre-B ALL CD34+ cells were i.v. transplanted to NSG mice. Both 104 and 105 cells resulted in the engraftment of the leukemia cells in limbs and cranium as judged by imaging after 6 weeks, and 103 cells engrafted after 13 weeks. When the 105 cells-transplanted mice were sacrificed after 14 weeks, the harvested peripheral blood, spleen (3–4×108cells/spleen) and bone marrow (5−10×106 cells/femur) displayed 2–3%, 51–55% and 75–81% of human CD34+CD19+ cells, respectively. Human CD34−CD19+ cells were 1–2%, 12–13% and 15–21%, respectively. Therefore, our stromal culture system supports leukemic stem cell/leukemia initiating cell proliferation and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo, and reveals the heterogeneity and hierarchical structure of human pre-B ALL cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides powerful tools to dissect the pathophysiology of human pre-B ALL, and to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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