A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Abstract Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders. Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs. To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies. Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. Figure 1 Figure 1. Disclosures Frank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

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PI3-Kinase Deletion Dysregulates Autophagy in HSCs and Promotes Myelodysplasia

Blood ◽

10.1182/blood-2021-150655 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 323-323

Author(s):

Kristina Ames ◽

Imit Kaur ◽

Shayda Hemmati ◽

Shira Glushakow-Smith ◽

Lindsay Meg Gurska ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Research Funding ◽

Akt Pathway ◽

Publicly Traded ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Autophagic Degradation

Abstract Myelodysplastic Syndrome (MDS) is a heterogeneous clonal malignancy arising in hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis, cytopenias, and the potential to progress to acute myeloid leukemia (AML). However, the perturbations in HSCs that lead to MDS initiation are poorly understood. It has been reported that HSCs are particularly dependent on autophagy for the maintenance of differentiation and self-renewal. We observed that, compared to healthy donor bone marrow hematopoietic stem and progenitor cells (HSPCs), MDS patient stem and progenitor cells (Lin-CD33-CD34+CD38-) have abnormal levels of autophagic degradation, as demonstrated by abnormal intracellular LC3II and P62 staining (Figure 1A). Autophagy is known to be regulated by the (PI3K)/AKT pathway, which transduces hematopoietic growth factor and cytokine signals in HSCs. PI3K/AKT is frequently activated in AML, but its role in MDS is less clear. Surprisingly, we found that CD34+ cells from a subset of MDS patients have upregulated expression of PTEN, the major negative regulator of the PI3K/AKT pathway, suggesting that PI3K/AKT may be downregulated in MDS stem cells. Therefore, we hypothesized that the Class IA PI3K isoforms (P110α, β, and δ) are required to maintain HSC differentiation and self-renewal. To understand the consequences of PI3K downregulation in HSCs, we generated a triple knockout (TKO) mouse model with conditional deletion of P110α and P110β in hematopoietic cells, and germline deletion of P110δ. Surprisingly, we found that PI3K deletion causes transplantable pancytopenia and decreased survival, despite the abnormal expansion of donor TKO HSCs (Figure 1 B,C). Consistent with this inefficient hematopoiesis, TKO bone marrow cells exhibited dysplastic features in multiple blood lineages and multiple chromosomal abnormalities (Figure 1 E,F), suggesting that PI3K inactivation in HSCs can promote MDS initiation. To determine whether impaired HSC differentiation in TKO mice could be due to dysregulated autophagy, we assessed autophagy in TKO HSCs by flow cytometry and immunofluorescence with the autophagosomal marker, LC3II. Our results showed that, compared to the WT controls, TKO HSCs have inefficient autophagic flux and decreased degradation of the cargo protein P62. We also discovered that TKO HSCs have significantly enlarged autophagic vesicles (Figure 1 G), and impaired fusion of autophagosomes with lysosomes, consistent with a marked defect in autophagic degradation. Treatment of TKO mice with two pharmacologic inducers of autophagy, rapamycin or metformin, improved HSC differentiation with an increase in Flk2+ MPPs (Figure 1 H), reduced dysplasia, and decreased the size of the TKO mutant clone in chimeric mice. Thus, our results uncover an important role for PI3K in regulating autophagy in HSCs to maintain the proper balance between self-renewal and differentiation. Our new mouse model of MDS will be a useful tool to study the mechanisms of MDs initiation. In addition, our findings open exciting avenues for future investigations of autophagy-inducing agents in MDS. Figure 1 Figure 1. Disclosures Verma: Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; BMS: Research Funding; GSK: Research Funding. Gritsman: iOnctura: Research Funding.

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Towards Improved Models of NPM-ALK Induced T-Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-115872 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1342-1342

Author(s):

Sophia Ehrenfeld ◽

Khalid Shoumariyeh ◽

Teresa Poggio ◽

Robin Khan ◽

Desiree Melanie Redhaber ◽

...

Keyword(s):

Stem Cells ◽

T Cell ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Human Disease ◽

Cell Lymphoma ◽

Specific Expression ◽

Hematopoietic Stem ◽

Stop Cassette

Abstract Time- and tissue-specific expression of transgenes is essential for the accurate representation of human disease in in vivo models. To improve flexibility but also fidelity of ALK+ ALCL models, we developed new approaches enabling lineage specific expression of NPM-ALK cDNAs, as well as a novel system for CRISPR/Cas induced Npm-Alk recombination. First, we have designed a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop-cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is rapidly achieved through retroviral infection and subsequent transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage specific promoter. To validate the model, we directed expression of NPM-ALK, the fusion oncogene present in anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre transgenic mice with a retroviral construct containing the Npm-Alk cDNA preceded by a translational stop cassette. The approach efficiently induced T-cell lymphomas within 12-16 weeks closely resembling the human disease including the expression of the ALCL hallmark antigen CD30. Since NPM-ALK overexpressed from a cDNA acts as a very strong oncogene transforming a range of cell types, we were interested to develop a more physiologic model based on chromosomal recombination, enabling NPM-ALK expression from the endogenous Npm promoter. To achieve this, we have designed guide RNAs (gRNAs) directed to either the intron between exon 4 and 5 for Npm1 on mouse chr. 11, or the intron between exons 19 and 20 for Alk on mouse chr. 17., enabling targeted translocation between the two chromosomes. For further analysis, the IL-3 dependent murine pro-B cell line Ba/F3 stably expressing the Cas9 recombinase was transduced with the respective gRNAs and subsequently grown in the absence of IL-3 to allow positive selection of cells transformed by productive t(11;17) NA recombination. A PCR reaction on genomic DNA using primers covering the translocation breakpoint resulted in a product and Sanger sequencing of the amplicon confirmed the intended recombination at the targeted genomic positions. The translocation was also detectable by fluorescence in-situ hybridization (FISH), and Western blot analysis demonstrated expression of a highly phosphorylated Npm-Alk fusion protein. To further probe for oncogene dependency, we treated Npm-Alk translocated cells and control cells with a specific Alk inhibitor, resulting in rapid cell depletion of the Npm-Alk translocated cells, but not controls. The described Cre/loxP-based system represents a versatile tool for the rapid functional analysis of gene function in a defined lineage or in a developmental stage in vivo, and faithfully recapitulates many features of ALCL. Furthermore, using Crispr/Cas to induce targeted double-strand breaks, we have been able to generate specific Npm-Alk translocations in murine cells, paving the way for novel models which may help to further define the initial pathogenetic event underlying lymphomagenesis in ALCL. Disclosures No relevant conflicts of interest to declare.

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Functional Characterization of Compound DDX41 Germline and Somatic R525H Mutations in the Development of Myeloid Malignancies

Blood ◽

10.1182/blood-2020-143318 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-22

Author(s):

Ayana Kon ◽

Masahiro Marshall Nakagawa ◽

Ryosaku Inagaki ◽

Keisuke Kataoka ◽

Hideki Makishima ◽

...

Keyword(s):

Stem Cells ◽

Research Funding ◽

Mutant Mice ◽

Loss Of Function ◽

Private Company ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Myeloid Malignancies ◽

Myeloid Neoplasms ◽

Cell Fractions

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 (Ddx41+/+), heterozygous Ddx41 KO (Ddx41+/-), heterozygous for the Ddx41 R525H mutation (Ddx41R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were reconstituted with BM from Ddx41R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF) with no development of myeloid neoplasms. However, about 20% of mice transplanted with BM derived from Ddx41R525H/- mice survived longer without showing BMF. These mice exhibited macrocytic anemia and increased platelet counts four months after tamoxifen-induction. In contrast, mice transplanted with BM from Ddx41+/- and Ddx41R525H/+ animals showed increased white blood cell counts compared to those with BM from Ddx41+/+ mice. In flow cytometry, Ddx41R525H/--derived BM-transplanted mice showed a significant increase in the number of long-term and short-term hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs) and granulocyte/macrophage lineage-restricted progenitors (GMPs), compared to those transplanted with BM from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Single cell RNA-seq of lineage negative cell fractions from these mice also revealed expanded stem cell fractions in mice transplanted with BM from Ddx41R525H/- mice, even though there was impaired formation of mature peripheral blood cells, which was suggestive of impaired HSPC differentiation. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41R525H/- mice-derived cells in PB was reduced compared to that of cells derived from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Transcriptome analysis of stem cells (Kit+Sca-1-Linlow cells) from different Ddx41 mutant mice revealed significant changes in gene expression and splicing patterns in many genes in stem cells from all the mutant mice, with larger changes for Ddx41R525H/- than Ddx41+/- or Ddx41 R525H/+ cells. Notably, Ddx41R525H/- cells exhibited a significant upregulation of genes involved in innate immunity, whereas there was a downregulation of genes related to RNA metabolism and ribosome biogenesis. Proteomics analysis confirmed the significant downregulation of ribosomal proteins in hematopoietic cells derived from Ddx41R525H/- mice. In summary, our results revealed an essential role of Ddx41 in normal hematopoiesis. While both heterozygous Ddx41 KO and heterozygous R525H knock-in did not develop myeloid neoplasm, compound biallelic loss-of function and R525 alleles led to a compromised function of hematopoietic stem cells, which was evident from reduced competitive repopulation capacity and impaired hematopoietic differentiation, where activated innate immunity and impaired ribosome functions may play important roles. Their roles in myeloid neoplasms need further evaluation. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Kataoka:Takeda Pharmaceutical Company: Research Funding; Asahi Genomics: Current equity holder in private company; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Otsuka Pharmaceutical: Research Funding. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company.

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Hematopoietic Stem Cells Fail to Trans-Differentiate into Smooth Muscle-Lineage Cells In Vivo.

Blood ◽

10.1182/blood.v106.11.1703.1703 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1703-1703

Author(s):

Takafumi Yokota ◽

Sanai Sato ◽

Yutaka Kawakami ◽

Yoshinori Nagai ◽

Jian-xing Ma ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Smooth Muscle ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Mesangial Cells ◽

Regulatory Sequence ◽

Gfp Expression ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Since bone marrow has been shown to contain cells capable of differentiating into various lineages, it is attracting considerable attention as a potential source for tissue regeneration. The plasticity of hematopoietic stem cells (HSC), however, remains controversial. While recent studies have suggested that HSC give rise to other tissue types, others argue against HSC plasticity. Because any tissue injury results in extensive recruitment of inflammatory cells, evidence for stem cell plasticity with non-specific green fluorescent protein (GFP) transgenic mice is often confounded by the results of adhesion and/or cell fusion by macrophages. Some reports using non-specific GFP transgenic mice demonstrated that HSC could contribute to the generation of smooth muscle type α-actin (αSMA)-expressing cells in injured arteries or renal gromeruli. We developed a new transgenic αSMA-GFP mouse with GFP coupled to an αSMA regulatory sequence. Bone marrow transplantation was then used to evaluate the potential of marked cells to generate donor type tissues in irradiation chimeras. There was a highly restricted pattern of GFP expression in the transgenics, marking vascular smooth muscle cells, pericytes and mesangial cells in the kidney, but no hematopoietic cells while the marrow contained infrequent cells positive for αSMA-GFP (0.02% of mononuclear cells). When the marrow cells were cultured, stromal cells with high GFP expression were abundantly produced in two weeks. Similarly cultured glomeruli generated many mesangial cells with high GFP expression. These characteristics, however, were not transferable to lethally irradiated (950rad) RAG-2 deficient mice, even when 1 x !07 transgenic marrow cells per recipient were infused via tail vein and the hematopoietic system was largely replaced by recipient cells. At 6 months after transplantation, no GFP+ cells were observed in either bone marrow or renal glomeruli of 20 RAG-2 deficient recipients. Furthermore, cultivation of their marrow or glomerular cells did not produce αSMA-GFP expressing cells. Our findings support earlier studies suggesting that the bone marrow microenvironment is difficult to transplant by standard methods and indicate that HSC are unlikely to give rise to αSMA expressing progeny.

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Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20180853 ◽

2021 ◽

Vol 218 (2) ◽

Cited By ~ 1

Author(s):

Eleni Louka ◽

Benjamin Povinelli ◽

Alba Rodriguez-Meira ◽

Gemma Buck ◽

Wei Xiong Wen ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Childhood Leukemia ◽

Clonal Evolution ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Ras Pathway ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin−CD34+CD38−CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a “first hit,” (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

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Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Blood ◽

10.1182/blood.v128.22.4107.4107 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4107-4107

Author(s):

Max Jan ◽

Florian Scherer ◽

David M. Kurtz ◽

Aaron M Newman ◽

Henning Stehr ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

B Cells ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Phylogenetic Trees ◽

Somatic Mutations ◽

Hematopoietic Stem ◽

Tumor Biopsies ◽

Myd88 L265p

Abstract Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

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Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Blood ◽

10.1182/blood-2019-129746 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4640-4640

Author(s):

Heng-Yi Liu ◽

Nezia Rahman ◽

Tzu-Ting Chiou ◽

Satiro N. De Oliveira

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Cell Lines ◽

Immune Cells ◽

Research Funding ◽

Humanized Mice ◽

Tumor Challenge ◽

Hematopoietic Stem

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

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NOTCH and CAR Signaling Control T Cell Lineage Commitment from Pluripotent Stem Cells

Blood ◽

10.1182/blood-2020-142894 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-30

Author(s):

Sjoukje van der Stegen ◽

Pieter Lindenbergh ◽

Roseanna Petrovic ◽

Benjamin Whitlock ◽

Raedun Clarke ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Cell Lineage ◽

Lineage Commitment ◽

Publicly Traded ◽

Car T Cell ◽

Car T ◽

Single Positive

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

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Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilia A mice with preexisting FVIII immunity

Blood ◽

10.1182/blood-2008-02-138214 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2713-2721 ◽

Cited By ~ 70

Author(s):

Qizhen Shi ◽

Scot A. Fahs ◽

David A. Wilcox ◽

Erin L. Kuether ◽

Patricia A. Morateck ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Factor Viii ◽

Hemophilia A ◽

Genetically Modified ◽

Hematopoietic Stem ◽

Inhibitory Antibodies ◽

Sublethal Irradiation

Abstract Although genetic induction of factor VIII (FVIII) expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of preexisting FVIII inhibitory antibodies to determine whether such antibodies would affect therapeutic engraftment. We generated a line of transgenic mice (2bF8) that express FVIII only in platelets using the platelet-specific αIIb promoter and bred this 2bF8 transgene into a FVIIInull background. Bone marrow (BM) from heterozygous 2bF8 transgenic (2bF8tg+/−) mice was transplanted into immunized FVIIInull mice after lethal or sublethal irradiation. After BM reconstitution, 85% of recipients survived tail clipping when the 1100-cGy (myeloablative) regimen was used, 85.7% of recipients survived when 660-cGy (nonmyeloablative) regimens were used, and 60% of recipients survived when the recipients were conditioned with 440 cGy. Our further studies showed that transplantation with 1% to 5% 2bF8tg+/− BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not negate engraftment of 2bF8 genetically modified hematopoietic stem cells, and transplantation of these hematopoietic stem cells can efficiently restore hemostasis to hemophilic mice with preexisting inhibitory antibodies under either myeloablative or nonmyeloablative regimens.

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Gpr44 Mediates the Selenium-Dependent Anti-Leukemic Effect in Acute Myeloid Leukemia

Current Developments in Nutrition ◽

10.1093/cdn/nzaa067_062 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1835-1835

Author(s):

Fenghua Qian ◽

Diwakar Tukaramrao ◽

Jiayan Zhou ◽

Nicole Palmiero ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Murine Model ◽

Myeloid Leukemia ◽

Hematopoietic Stem ◽

Cell Counts ◽

Pparγ Agonist ◽

Acute Myeloid

Abstract Objectives The relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia stem cells (LSCs) that are not targeted by existing therapies. LSCs show sensitivity to endogenous cyclopentenone prostaglandin J (CyPG) metabolites that are increased by dietary trace element selenium (Se), which is significantly decreased in AML patients. We investigated the anti-leukemic effect of Se supplementation in AML via mechanisms involving the activation of the membrane-bound G-protein coupled receptor 44 (Gpr44) and the intracellular receptor, peroxisome proliferator-activated receptor gamma (PPARγ), by endogenous CyPGs. Methods A murine model of AML generated by transplantation of hematopoietic stem cells (HSCs- WT or Gpr44−/−) expressing human MLL-AF9 fusion oncoprotein, in the following experiments: To investigate the effect of Se supplementation on the outcome of AML, donor mice were maintained on either Se-adequate (Se-A; 0.08–0.1 ppm Se) or Se-supplemented (Se-S; 0.4 ppm Se) diets. Complete cell counts in peripheral blood were analyzed by hemavet. LSCs in bone marrow and spleen were analyzed by flow cytometry. To determine the role of Gpr44 activation in AML, mice were treated with Gpr44 agonists, CyPGs. LSCs in bone marrow and spleen were analyzed. Mice transplanted with Gpr44−/- AML cells were compared with mice transplanted with wild type AML cells and the progression of the disease was followed as above. To determine the role of PPARγ activation in AML, PPARγ agonist (Rosiglitazone, 6 mg/kg, i.p, 14 d) and antagonist (GW9662, 1 mg/kg, i.p. once every other day, 7 injections) were applied to Se-S mice transplanted with Gpr44−/- AML cells and disease progression was followed. Results Se supplementation at supraphysiological levels alleviated the disease via the elimination of LSCs in a murine model of AML. CyPGs induced by Se supplementation mediate the apoptosis in LSCs via the activation of Gpr44 and PPARγ. Conclusions Endogenous CyPGs produced upon supplementation with Se at supraphysiological levels improved the outcome of AML by targeting LSCs to apoptosis via the activation of two receptors, Gpr44 and PPARg. Funding Sources NIH DK 07,7152; CA 175,576; CA 162,665. Office of Dietary Supplements, USDA Hatch funds PEN04605, Accession # 1,010,021 (KSP, RFP).

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