Functional Analysis of the Subclonal Architecture of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) at a Single Cell Level

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3778-3778
Author(s):  
Bartosch Wojcik ◽  
Fabian Lang ◽  
Susanne Badura ◽  
Anja Vogel ◽  
Tamara Tesanovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Research Funding ◽  
Cell Tracking ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Cd38 Expression ◽  
Cell Fractions

Abstract Introduction: Transforming events in B-lineage (ALL) occur primarily at the level of committed progenitor cells, but the phenotype, frequency and hierarchical organization of leukemia-initiating cells (LICs) are controversial. Pronounced clonal heterogeneity in ALL and the lack of preclinical models impede functional analysis of LICs and dissecting the clonal architecture of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Aims and methods: To identify cellular features associated with cell fate and decisions, engraftment potential, growth kinetics and resistance to therapy of BCP-ALL LICs at the clonal level, we generated multiple single-cell-derived clones from patient derived-long-term cultures (PD-LTCs) of human BCP-ALL. This in vitro ALL-LTC model allows prolonged serum-, cytokine-, and stroma-free culture of patient-derived BCP-ALL cells that are largely stable in terms of cytogenetic and immunophenotypic features for up to 6 months [Nijmeijer B et. al, Exp Hematol. 2009;37:376; Badura S et al., PLoS One. 2013; 8:e80070] Three ALL-LTCs were selected on the basis of different patterns of surface marker expression and cytogenetics, i.e. Ph+ ALL (LTCs PH and BV; common-ALL) and BCR-ABL negative ALL (LTC CR; pre B-ALL), respectively. Results: Clonogenic growth in semisolid medium ranged from 0.25% to 8% for these ALL-LTCs (CR 8%, PH 0.45% and BV 0.25%), consistent with functional diversity within the bulk as well as the immunophenotypically defined subsets. To analyze these subpopulations at the clonal level, we isolated leukemic cell subsets by cell sorting on the basis of CD20, CD34 and CD38 expression, and then generated hundreds of clones by expanding single-cell sorted subpopulations. All sorted cell fractions from all three ALL-LTCs were capable of initiating long-term growth in vitro. In limiting dilution cultures, 1% to 5% of single sorted cells derived from these cell fractions gave rise to prolonged leukemic cell growth. To evaluate the leukemia-initiating capacity of the clonal ALL subpopulations in vivo, we examined engraftment and outgrowth kinetics of different ALL clones in a xenograft model with sublethally irradiated NSG mice. Individual clones derived from different sorted subpopulations displayed pronounced differences in engraftment potential and aggressiveness based on analyses of blood, bone marrow and spleen. This ranged from rapid appearance of ALL and death within 70-80 days (clone PH 20), to complete lack of leukemic outgrowth 155 days after transplantation (clone PH clone 14). The other four clones derived from this ALL (PH) displayed intermediate engraftment and outgrowth kinetics. Notably, leukemogenic properties were not associated with a distinct surface marker profile based in CD20, CD34 and CD38 expression. A similar heterogeneity of leukemogenic potential was determined with clonal subpopulations from the LTC BV. To assess the hierarchical relationship between different clones and the association with LIC capacity, we examined the pattern of immunoglobulin VDJ-rearrangement of different clones. The Ig rearrangement patterns confirm that the clones represent distinct clonal populations derived from the original polyclonal LTC, but we observed no hierarchical relationship with respect to the biological properties of different clones. Further functional evaluation of these subclones was performed by videomicroscopy-based single cell tracking that allows analysis of individual cells and their progeny over many cell divisions. We observed concordance between cell cycle length and cell death events as determined by single cell tracking and the engraftment potential and aggressiveness of LTCs derived single cell clones in vivo. To elucidate the molecular basis for the biologic differences between the clonal ALL populations, we compared highly and poorly leukemogenic clones by SILAC based proteomics. Only 5% of 6500 proteins measured in clones differing in their leukemia-initiating potential were differentially regulated, and these proteins could be assigned to a limited number of pathways. In conclusion, comparative evaluation of clonal lymphoblast populations generated from patient-derived ALL long-term cultures by combined in vitro analysis, single cell videomicroscopy, xenotransplantation and proteomics is a promising approach to identify specific markers and therapeutic strategies that target LIC in ALL. Disclosures Wojcik: Novartis: Research Funding, Travel, Accommodation Other; SAnofi Aventis: Consultancy, Travel, Accommodation, Travel, Accommodation Other. Lang:Novartis: Research Funding, Travel, Accommodation Other. Brüggemann:Amgen Inc.: Consultancy, Research Funding.

scholarly journals A Predictive Endothelial-Leukemia Pre-Clinical Platform to Uncover Drug Vulnerabilities for Personalized Treatments

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 704-704
Author(s):  
Luca Vincenzo Cappelli ◽  
Danilo Fiore ◽  
Jude M Phillip ◽  
Liron Yoffe ◽  
Filomena Di Giacomo ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Single Cell ◽  
Research Funding ◽  
Drug Response ◽  
Lymphoblastic Leukemia ◽  
Principal Component ◽  
Study Drug ◽  
Patient Specific

Abstract Background. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with few innovative treatment options. This is also contributed by the lack of models capable of capturing the complexity of the tumor and its microenvironment. Aims. To identify patient-specific vulnerabilities and novel therapeutic strategies in T-ALL and interrogate the mechanisms of the crosstalk between leukemic and stromal elements. Methods. We established a drug-testing platform using patient-derived-tumor-xenografts (PDTX) and a mixed-culture approach using E4ORF1-transduced endothelial cells (ECs) (Seandel M et al, PNAS 2008) to overcome host-mediated chemoresistance. We performed functional experiments using total and single-cell RNA sequencing. Results. First, we established a battery of 22 T-ALL PDTX models that matched both phenotypically (immune-histochemistry, flow cytometry) and genotypically (TCR rearrangement, transcriptome) with the primary patients' samples. We then challenged these models (n=14 samples belonging to different PDTX and serial passages within each model) with a library of compounds (n=433) targeting redundant proteins (n=634). Unsupervised clustering and Principal Component Analysis (PCA) demonstrated two clusters of T-ALL samples based on differential drug susceptibility. We could at least partially correlate these differences to specific transcriptomic signatures predictive of drug response (Figure 1A). We then defined a group of pan-active compounds across all models (n=40), which we validated using an independent screening with/without ECs (Figure 1B). We found that ECs counteracted the activity of selected compounds (i.e. TSA, THZ1 and MLN2238). By PCA, we observed distinct response profiles based on different T-ALL models. We vectorized the EC-rescue and found that the direction was the same across all 3 models tested, indicating that it relied on similar mechanisms regardless of model identity. Based on the known role of IGF1-IGFR1 as a supportive EC-rescue axis (Medyouf H et al, J Exp Med 2011), we performed the same screening with/without recombinant IGFBP-7 (500 ng/mL), a decoy IGF1 molecule. Remarkably, IGFBP-7 completely or partially abrogated the EC-mediated rescue of selected compounds [enzastaurin (PKC-β inhibitor), SC144 (GP130 inhibitor), CHIR124 (Chk1 inhibitor) and YM155 (Survivin inhibitor)] (Figure 1B). Drugs not rescued by ECs (n=30) were considered positive hits and 5 of them (ruxolitinib, tofacitinib, panobinostat, bortezomib, irinotecan) ultimately proved to be effective in vivo in randomized pre-clinical trials either alone or in combination (Figure 1C). Our stepwise endothelial-leukemia platform led to the discovery of "public" and "private" vulnerabilities and the proof-of-principle of prediction-guided in vivo pre-clinical trials. We propose a list of compounds that could be readily translated into T-ALL clinical trials (Figure 1D). We finally proved the validity of our platform using other disease models (i.e. B and T-lymphoma PDTXs). Mechanistically, at single-cell resolution, in vitro interacting T-ALL cells and ECs underwent reciprocal transcriptome changes, with T-ALL shifting towards stemness/undifferentiation and ECs towards tumor-ECs (TECs) phenotypes. Furthermore, in vitro EC-educated T-ALL cells mimicked distinct T-ALL subsets of the leukemic spleen of corresponding PDTX mice (Figure 1E). Conclusions. These data demonstrate that our EC-T-ALL culture system simulates in vivo conditions, offering a robust platform to study drug response, leukemia-host interactions and cell plasticity. This approach will improve the pre-clinical predictability of novel drugs/combinations for T-ALL, as well as for other hematologic malignancies, and propel the development of patient-tailored treatments. Figure 1 Figure 1. Disclosures Melnick: Janssen Pharmaceuticals: Research Funding; Sanofi: Research Funding; Daiichi Sankyo: Research Funding; Epizyme: Consultancy; Constellation: Consultancy; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Elemento: AstraZeneca: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Owkin: Consultancy, Other: Current equity holder; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Janssen: Research Funding. Chiaretti: amgen: Consultancy; pfizer: Consultancy; novartis: Consultancy; Incyte: Consultancy. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

scholarly journals Long-term stable hematopoietic chimerism following marrow transplantation for acute lymphoblastic leukemia: a case report with in vitro marrow culture studies

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 869-872 ◽  
Author(s):  
JW Singer ◽  
A Keating ◽  
R Ramberg ◽  
R McGuffin ◽  
JE Sanders ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Mononuclear Cells ◽  
Lymphoblastic Leukemia ◽  
Host Cells ◽  
Marrow Graft ◽  
Peripheral Blood Mononuclear ◽  
Hematopoietic Chimerism

Abstract This article describes the course of a patient who received an allogeneic marrow graft from his HLA-identical sister for acute lymphoblastic leukemia in second remission. In the second month after grafting, marrow aspirates showed the presence of 7%-10% lymphoblasts. In addition, cytogenetic examination indicated the persistence of host cells. Thereafter, the patient had morphologically normal marrow examinations, with no evidence for recurrent leukemia. In addition, stable hematopoietic chimerism in both the lymphoid and myeloid cell lines has persisted for over 5 yr. Between 20% and 50% of phytohemagglutinin-stimulated peripheral blood mononuclear cells were host-derived on repeated studies. A marrow sample 4 yr after transplantation was established in long-term culture and produced 2% host granulocyte-macrophage colonies at its inception, but 24% host colonies by week 4. Despite this persistent chimerism, no in vitro or in vivo abnormalities of hematopoiesis have been detected.

Metastasis Suppressor 1 Is Downregulated in CML Stem Cells and Overexpression Impairs Early Leukemic Cell Propagation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2776-2776
Author(s):  
Mirle Schemionek ◽  
Ulrich Steidl ◽  
Albrecht Schwab ◽  
Daniel G Tenen ◽  
Copland Mhairi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Research Funding ◽  
Leukemic Cell ◽  
Metastasis Suppressor ◽  
Advisory Committees ◽  
Abl Kinase ◽  
Metastasis Suppressor 1 ◽  
Cell Propagation

Abstract Abstract 2776 The implementation of Bcr-Abl tyrosine kinase inhibitors (TKIs) has greatly improved the outcome of patients with chronic myeloid leukemia (CML). However, discontinuation of TKI therapy often results in relapse suggesting that leukemic stem cells (LSCs) survive despite treatment. More detailed investigations utilizing patient samples and murine CML models have confirmed that the leukemia-initiating cell population is usually not eradicated by inhibiting Bcr-Abl activity and that this is due to a lack of oncogene addiction of LSCs, showing that further research is required aiming to fully understand LSC biology. To identify new Bcr-Abl targets that are involved in LSC persistence, we performed a microarray analysis of the leukemia-initiating cell population in an inducible transgenic SCLtTAxBcr-Abl CML mouse model in which we had previously shown that these cells are not oncogene-addicted (Schemionek et al., BLOOD 2010; Hamilton et al., BLOOD 2012). One of the most downregulated genes in CML vs. normal stem cells was Metastasis Suppressor 1 (Mtss1/MIM). Although the multidomain protein Mtss1 may be involved in carcinogenesis of several solid tumors, its exact physiological role is still unknown. Current findings suggest that Mtss1 interacts with multiple partners and is involved in various signalling pathways that regulate actin dynamics and cell motility. Interestingly, Rac and Src are Mtss1 interacting partners, and both proteins are known to be involved in Bcr-Abl mediated leukemogenesis. We have previously shown that Mtss1 is downregulated in mouse LSCs and demonstrated that this is a Bcr-Abl kinase mediated effect in various human and murine CML cell lines. Moreover, we have shown that Mtss1 overexpression in 32D-Bcr-Abl cells induces a defect in Bcr-Abl mediated migration in vitro and reduces the potential to form solid tumors in vivo. Here we show that Bcr-Abl kinase-dependent regulation of Mtss1 expression was also evident in mononuclear cells and CD34+ progenitor cells from patients with CML upon IM or dasatinib treatment in vitro. Moreover, cells from IM-treated patients with chronic phase CML showed elevated Mtss1 expression levels within one to three weeks of treatment. Increasing Mtss1 expression upon 5-aza-2′-deoxycytidine-treatment of K562 and 32D-Bcr-Abl cells suggested that methylation might be involved in Mtss1 regulation. To determine a potential leukemia suppressing effect of Mtss1 overexpression, we performed colony assays using lineage negative SCLtTAxBcr-Abl (dtg) bone marrow (BM) cells that had been retrovirally infected to overexpress Mtss1 (dtg::Mtss1) or empty-vector (dtg::ev). Successfully transduced BM cells were FACS-sorted via GFP-expression, encoded by the retroviral vector. Mtss1 overexpression led to a 2.3-fold decrease in CFU numbers. In a second set of experiments we transplanted 1.3×105 GFP-FACS-sorted dtg::Mtss1 or dtg::ev cells into 9 Gy-irradiated recipients. While dtg::ev recipients contained 66% (+/−8%) of GFP-positive cells in the BM, these cells were decreased in dtg::Mtss1 transplanted mice to 23% (+/−21%), 12 days after transplantation. A similar effect was evident in the spleen [dtg::ev recipients: 90% (+/− 3%) versus dtg::Mtss1 recipients 59% (+/−20%)] suggesting that Mtss1 confers a disadvantage to Bcr-Abl positive BM cells in the early steps of leukemic cell propagation, compared to Bcr-Abl cells alone. Since the multidomain Mtss1 protein contains a putative Abl-SH2-binding site, we performed co-immunoprecipitations using 32D-Bcr-Abl-Flag-Mtss1 cells. These experiments showed that both proteins were direct binding partners and that Mtss1 was not phosphorylated by Bcr-Abl. Taken together, our data show that Mtss1 is downregulated via a Bcr-Abl kinase mediated mechanism and this might involve methylation. Moreover, additional inhibition of Mtss1 activity might be mediated through direct binding by Bcr-Abl. Forced expression of the potential tumor suppressor in CML stem and progenitor cells reduces leukemic cell propagation in vivo and may thus provide a rationale to contribute to LSC elimination in patients with CML. Disclosures: Mhairi: BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals CD7+, CD4-, CD8- acute leukemia: a syndrome of malignant pluripotent lymphohematopoietic cells

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 381-390
Author(s):  
J Kurtzberg ◽  
TA Waldmann ◽  
MP Davey ◽  
SH Bigner ◽  
JO Moore ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Therapeutic Approaches ◽  
Myeloid Leukemias ◽  
Cell Gene ◽  
In Vitro Data

Following our initial observation of in vivo conversion of CD7+, CD4-, CD8- acute lymphoblastic leukemia (ALL) cells from lymphoid to myeloid lineages (Proc Natl Acad Sci (USA) 81:253, 1984) we have studied eight additional cases of ALL with this leukemic cell phenotype. The CD7+, CD4-, CD8- phenotype was associated with a distinct clinical entity with those affected predominantly male (either less than 35 years or greater than 65 years of age), with frequent mediastinal and/or thymic masses, skin and CNS disease, high peripheral WBC counts, and bone marrow blasts that were morphologically L1 or not ascribable to a specific lineage. These patients did not respond to conventional chemotherapeutic regimens for either acute lymphoid or myeloid leukemias. No common karyotype or T-cell gene rearrangement pattern could be defined. Importantly, seven of eight patient's leukemic cells studied were capable of multilineage (myeloid, erythroid, monocytoid, megakaryocytoid, and lymphoid) differentiation in vitro. Data is presented suggesting that CD7+, CD4-, CD8- leukemias, in many instances, are leukemias of immature hematopoietic cells. The development of novel therapeutic approaches to this form of leukemia will be necessary to alter its poor prognosis.

scholarly journals AVA4746, an Orally Available, Clinical Grade Antagonist of Integrin Alpha 4, Sensitizes Pre-B Cell Acute Lymphoblastic Leukemia to Chemotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2765-2765 ◽  
Author(s):  
Yongsheng Ruan ◽  
Eun Ji Gang ◽  
Hye-Na Kim ◽  
Chintan Parekh ◽  
Hisham Abdel-Azim ◽  
...  
Keyword(s):  
Overall Survival ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Metabolic Activity ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Drug Resistant ◽  
Support Research

Abstract Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL. Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis. Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P<0.05). There was no significant difference in survival between the PBS control group (n=5) and the AVA4746 mono-treatment group (n=5) (MST=38days vs MST= 38days). Conclusion. We have identified α4 as a central adhesion molecule in CAM-DR of ALL and have shown that AVA-4746, an orally available and specific α4 antagonist, which has been safely used in clinical studies, downregulates α4 in primary ALL and functionally de-adheres them from VCAM-1. Critically, we demonstrated that inhibition of α4 in combination with standard chemotherapy can prolong the survival of NSG mice bearing pre-B ALL. These data support further study of inhibition of α4 using AVA4746 as a novel strategy to treat drug resistant B lineage ALL. Disclosures Bhojwani: Amgen: Other: Blinatumumab global pediatric advisory board 2015. Wayne:Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Kite Pharma: Honoraria, Other: Travel support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Kim:Antisense Therapeutics Ltd: Patents & Royalties.

scholarly journals Results of CoALL 07-03 study childhood ALL based on combined risk assessment by in vivo and in vitro pharmacosensitivity

2019 ◽  
Vol 3 (22) ◽  
pp. 3688-3699 ◽  
Author(s):  
Franziska Schramm ◽  
Udo zur Stadt ◽  
Martin Zimmermann ◽  
Norbert Jorch ◽  
Arnulf Pekrun ◽  
...  
Keyword(s):  
Drug Testing ◽  
Drug Response ◽  
Lymphoblastic Leukemia ◽  
Term Outcome ◽  
Childhood All ◽  
Long Term Outcome ◽  
Key Points

Key Points Report of the long-term outcome of children with acute lymphoblastic leukemia upon risk-adapted therapy accrued in trial CoALL 07-03. Lack of correlation between in vitro and in vivo drug response as well as a lower predictive value of in vitro drug testing.

scholarly journals Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1322-1322
Author(s):  
Manabu Kusakabe ◽  
Ann Chong Sun ◽  
Kateryna Tyshchenko ◽  
Rachel Wong ◽  
Aastha Nanda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

scholarly journals Two-Year Landmark Survival Analysis after Allogeneic Hematopoietic Cell Transplantation in Acute Lymphoblastic Leukemia: An Analysis from the ALWP of the EBMT

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1822-1822
Author(s):  
Vivek Patel ◽  
Myriam Labopin ◽  
Thomas Schroeder ◽  
Igor Wolfgang Blau ◽  
Lars Klingen Gjaerde ◽  
...  
Keyword(s):  
Survival Analysis ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Chronic Gvhd ◽  
Long Term Outcomes ◽  
Term Survival ◽  
Free Survival ◽  
Late Mortality

Abstract Background Long-term survival and late mortality risk compared to general population for patients (pts) who underwent an allogeneic hematopoietic cell transplant (HCT) is unknown. We analyzed long-term outcomes of 2-year (yr) HCT survivors with acute lymphoblastic leukemia (ALL). Methods Adult pts with ALL who were alive and relapse-free at 2 yrs after first HCT from 2005-2012 were included. We excluded patients who had a cord blood transplant and ex vivo T cell depletion (TCD). Relative survival analysis was used to estimate HCT-related crude mortality taking into account for background population mortality rates of the general population, matched for age, sex, and country in the year of HCT (www.mortality.org). Results A total of 2701 pts were included with a median follow up interval of 99 months and median age of 34 (range 18 - 73.5) yrs. The majority (78.6%) of pts were in 1 st complete remission (CR1) with undetectable MRD (68.3%). There were similar numbers of matched sibling donor (MSD) (43.7%) and unrelated donor transplants (MUD) (53.2%). Most pts received myeloablative conditioning (MAC) (86.5%) and peripheral blood (PB) grafts (75.7%) without in vivo TCD (55.7%). The 10-yr probability for overall survival (OS) and leukemia-free survival (LFS) was 81.3% and 78.2%, respectively. Cumulative incidence of disease relapse and non-relapse mortality (NRM) at 10 years was 9.9% and 11.9%, respectively. The probability of chronic GVHD-relapse-free survival (cGRFS) at 10-yrs was 73.3% (Figure 1). Relapsed ALL and chronic GVHD were common causes of late mortality accounting for 33.9% and 29% of reported deaths, respectively, followed by infection and secondary malignancy. For patients transplanted in countries with available mortality data (92% of patients in our cohort), the probability of dying from another cause is negligible at 1.5% compared to the probability of dying from HCT (16.8%) 10 years after HCT (Figure 1F). Conclusions In a large registry-based study, we showed excellent long-term survival of 81.3% at 10-yr among the 2-yr survivors of HCT for ALL. There was no difference in long term outcomes with respect to conditioning intensity, but utilization of BM graft and in vivo TCD resulted in lower NRM, and better OS. Long-term mortality risk among HCT survivors remains significantly higher than expected for the general age-matched population. Figure 1 Figure 1. Disclosures Labopin: Jazz Pharmaceuticals: Honoraria. Schroeder: Celgene: Honoraria, Other: Travel support, Research Funding. Bethge: Miltenyi Biotec: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Kite-Gilead: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Nicholson: Pfizer: Consultancy; BMS/Celgene: Consultancy; Kite, a Gilead Company: Other: Conference fees, Speakers Bureau; Novartis: Consultancy, Other: Conference fees. Giebel: Janssen: Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Peric: Therakos, Servier, MSD, Astellas, Novartis, Abbvie, Pfizer: Honoraria. Dholaria: Janssen: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Jazz: Speakers Bureau; MEI: Research Funding; Angiocrine: Research Funding; Poseida: Research Funding; Celgene: Speakers Bureau. Mohty: Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Adaptive Biotechnologies: Honoraria.

scholarly journals Integrated Single Cell Transcriptomics Defines an Engineered Niche Supporting Hematopoietic Stem Cell Development Ex Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3699-3699
Author(s):  
Brandon Hadland ◽  
Barbara Varnum-Finney ◽  
Stacey Dozono ◽  
Tessa Dignum ◽  
Cynthia Nourigat-Mckay ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Research Funding ◽  
Signal Pathways ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Cell Rna Sequencing ◽  
Equity Ownership

During embryonic development, hematopoietic stem cells (HSC) arise from hemogenic endothelial cells (HEC) within arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros) region, in a process referred to as the endothelial to hematopoietic transition (EHT). Although numerous signal pathways have been implicated in EHT, the precise combination of niche-derived signals required to support the generation and self-renewal of functional, long-term engrafting HSC remains poorly defined. To elucidate the niche signals regulating HSC emergence, we used single cell RNA-sequencing to simultaneously analyze the global transcriptional profiles of HEC during their transition to HSC and the AGM-derived endothelial cell stroma (AGM-EC) that supports the generation and expansion of functional HSC. Trajectory analysis of single cell transcriptomes enabled reconstruction of EHT in pseudotime, revealing dynamics of gene expression, including genes encoding cell surface receptors and downstream pathways, during the process of HSC genesis and self-renewal in vivo and in vitro. Transcriptional profiles of niche AGM-EC enabled identification of corresponding ligands which serve to activate these receptors during HSC generation. We integrated this knowledge to engineer a stromal cell-free niche for generation of engrafting HSC from hemogenic precursors in vitro. Specifically, we defined serum-free conditions combining immobilized Notch1 and Notch2-specific antibodies to activate Notch receptors, recombinant VCAM1-Fc chimera or fibronectin fragment to bind VLA-4 integrin, recombinant interleukin-3, stem cell factor, thrombopoietin, and CXCL12 to activate their respective cytokine/chemokine receptors, and small molecule inhibition of TGF-β Receptor 1. We demonstrated that this engineered niche is sufficient to support the generation of functional HSC, as measured by long-term (24 week) multilineage engraftment after transplantation to immune-competent, lethally irradiated adult recipient mice, following culture of hemogenic precursors isolated from E9.5 to E10.5 murine embryos. The observed efficiency of generating long-term engrafting HSC, particularly from precursors derived from early embryonic stages before E10, was lower in engineered conditions compared with AGM-EC stroma, suggesting additional niche signal factors remain to be defined to optimally support HSC maturation and self-renewal in the engineered niche. Single cell RNA-sequencing of hematopoietic progeny generated following culture in the engineered niche demonstrated the formation of populations with transcriptional signatures of HSC, as well as multipotent and lineage-specific progenitors, comparable to those generated following co-culture with niche AGM-EC stroma. However, we observed relative overexpression of Notch target genes promoting early T-lymphoid fate in cells generated from the engineered niche compared to those from AGM-EC stroma. Incorporating stage-specific attenuation of Notch1 receptor activation with soluble Notch1 blocking antibody during culture was sufficient to limit markers of early T-cell precursors, suggesting that temporal titration of Notch signal activation could be used to further modulate HSC and T-lymphoid output in the engineered niche. Altogether, these studies enhance our understanding of the core signal pathways necessary for the embryonic development of functional HSC, with the potential to advance in vitro engineering of therapeutically relevant pluripotent stem cell-derived HSC in stromal cell-free culture. Disclosures Bernstein: Lyell Immunopharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Nohla Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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