scholarly journals Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies

2019 ◽  
pp. 201819992 ◽  
Author(s):  
Florence Borot ◽  
Hui Wang ◽  
Yan Ma ◽  
Toghrul Jafarov ◽  
Azra Raza ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Residual Disease ◽  
Immune Therapy ◽  
Leukemic Cells ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Genetic Ablation ◽  
Targeted Immunotherapy

Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn’t impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

scholarly journals High levels of interleukin 10 production in vivo are associated with tolerance in SCID patients transplanted with HLA mismatched hematopoietic stem cells.

1994 ◽  
Vol 179 (2) ◽  
pp. 493-502 ◽  
Author(s):  
R Bacchetta ◽  
M Bigler ◽  
J L Touraine ◽  
R Parkman ◽  
P A Tovo ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Mrna Expression ◽  
Fetal Liver ◽  
Hla Antigens ◽  
Interleukin 10 ◽  
Hematopoietic Stem

Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.

Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2424-2424
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Third Party ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

scholarly journals Human MAIT cells are devoid of alloreactive potential: prompting their use as universal cells for adoptive immune therapy

2021 ◽  
Author(s):  
Marie Tourret ◽  
Nana Talvard-Balland ◽  
Marion Lambert ◽  
Ghada Ben Youssef ◽  
Mathieu F. Chevalier ◽  
...  
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Adoptive Therapy ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Mait Cells ◽  
Microbial Defense

ABSTRACTBackgroundMucosal associated invariant T (MAIT) cells are semi-invariant T cells that recognize microbial antigens presented by the highly conserved MR1 molecule. MAIT cells are predominantly localized in the liver and barrier tissues and are potent effectors of anti - microbial defense. MAIT cells are very few at birth and accumulate gradually over a period of about 6 years during infancy. The cytotoxic potential of MAIT cells, as well as their newly described regulatory and tissue repair functions, open the possibility of exploiting their properties in adoptive therapy. A prerequisite for their use as “universal” cells would be a lack of alloreactive potential, which remains to be demonstrated.MethodsWe used ex vivo, in vitro and in vivo models to determine if human MAIT cells contribute to allogeneic responses.ResultsWe show that recovery of MAIT cells after allogeneic hematopoietic stem cell transplantation recapitulates their slow physiological expansion in early childhood, independent of recovery of conventional T cells. In vitro, signals provided by allogeneic cells and cytokines do not induce sustained MAIT cell proliferation. In vivo, human MAIT cells do not expand nor accumulate in tissues in a model of T-cell mediated xenogeneic graft-versus-host disease (GVHD) in immunodeficient mice.ConclusionsAltogether, these results provide evidence that MAIT cells are devoid of alloreactive potential and pave the way for harnessing their translational potential in universal adoptive therapy overcoming barriers of HLA disparity.

ZFN-Driven Gene Editing Prevents HLA-A Expression On Hematopoietic Stem Cells -Improving The Chance Of Finding An HLA-Matched Donor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1655-1655
Author(s):  
Hiroki Torikai ◽  
Tiejuan Mi ◽  
Sonny O Ang ◽  
Loren Gragert ◽  
Martin Maiers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Hematopoietic Stem Cells ◽  
Donor Pool ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Immune Mediated ◽  
Matched Donor

Abstract Hematopoietic stem cells (HSCs) are administered (i) to restore hematopoiesis and immunity in the course of hematopoietic stem-cell transplantation (HSCT), (ii) as a replacement for inherited blood disorders and bone marrow failure, (iii) to regenerate cells of alternative lineages for restorative medicine, and (iv) as a source for generating specific hematopoietic cells (e.g., T cells, NK cells, and dendritic cells). However, the widespread application of allogeneic HSCs for humans is hampered by their immune-mediated destruction by host T cells recognizing mismatched HLA or by HLA-specific antibodies. Despite pre-banking umbilical cord blood (UCB) units and access to adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product is challenging for many recipients, especially those from ethnic minorities who are under-represented in the donor pool. The available donor pool would be markedly increased if donor HSCs were edited to eliminate expression of the HLA-A locus. Indeed, modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when matched for HLA-B, C and DR, instead of HLA-A, B, C and DR. We have previously shown that engineered zinc finger nucleases (ZFNs) can disrupt HLA-A expression in genetically edited T cells (Blood 2013). To extend this proof-of-concept to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus. CD34+lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB. Electro-transfer of in vitro transcribed mRNA encoding the HLA-A-specific ZFN generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1). As expected, SR1 treatment maintained greater numbers of CD34+ cells (also CD34posCD38neg) in culture compared to controls. DNA sequence analysis revealed that HLA-Aneg HSCs encode the expected nucleotide changes at the ZFN target site. An in vivo engraftment assay, using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, demonstrated that HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg hematopoietic cells (Figure). Thus, disruption of HLA-A expression in HSCs provides an appealing approach to increasing the chances for of finding HLA-matched donors and may broaden the clinical application of allogeneic HSCT. Furthermore, the ability to genetically edit HSCs has implications for (i) preventing immune-mediated recognition of HLA-disparate HSC and (ii) preventing immune mediated recognition of self-antigens. Engraftment of HLA-A2neg HSCs was evaluated in vivo. Data shown are flow-cytometry analysis of bone marrow obtained from NSG mice 16 weeks after HSC injection. HSC engraftment and HLA-A2 expression in NSG mice injected with un-modified HSCs (left panel) and HSCs treated with the HLA-A specific ZFNs (right panel) are shown. Data are gated on human CD45 positive cells. Figure Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Figure. Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

scholarly journals Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1333-1341 ◽  
Author(s):  
P Tiberghien ◽  
CW Reynolds ◽  
J Keller ◽  
S Spence ◽  
M Deschaseaux ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Herpes Simplex ◽  
Marrow Transplantation ◽  
Ex Vivo ◽  
Retroviral Vector ◽  
Hematopoietic Stem

Abstract Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus- leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV- induced T-cell depletion in allogeneic BMT recipients.

scholarly journals Pre-Clinical Development of Gene Modification of Hematopoietic Stem Cells with Chimeric Antigen Receptors for Cancer Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4430-4430
Author(s):  
Laurel Christine Truscott ◽  
Sarah Larson ◽  
Amie Patel ◽  
Roy L. Kao ◽  
Satiro N. De Oliveira
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
T Cells ◽  
Immune Cells ◽  
Suicide Gene ◽  
Antigen Specificity ◽  
Hematopoietic Stem ◽  
Cancer Activity

Abstract Background: Patients with refractory or recurrent B-lineage hematological malignancies have less than 50% of chance of cure, despite intensive therapy. Innovative approaches are needed to decrease their morbidity and mortality. Chimeric Antigen Receptors (CAR) successfully engineer antigen specificity in immune cells, with clinical trials currently being conducted using ex vivo expanded gene-modified mature T cells. Results from preclinical studies and clinical trials show that effector cells usually have transient in vivo persistence that could significantly limit clinical efficacy and allow tumor recurrence. Building upon the standard of care and seeking an approach that could foster persistence of the CAR-modified cells, we have published studies using anti-CD19 CAR-modified human hematopoietic stem cells (HSC) engrafted in NSG immunodeficient mice. We hypothesize that modification of HSC with CD19-specific CAR will generate persistent multi-lineage anti-tumor activity for immunotherapy of CD19+ hematological malignancies. To increase the safety of the modification of HSC, a suicide gene can be inserted into the vector to eradicate the modified cells in the setting of toxicity. Thorough evaluation of this approach in relevant study models is required for advancement to clinical trials. Significance: This approach is untested in clinical translation to this date, and implies harnessing a patient's own HSC to create a whole self-renewing immune system directed to destroy cancer, a concept that can be applied to different cancers just by adjusting the target specificity. The prospect of modifying autologous cells to enhance graft-versus-cancer activity bears the possibility of decreased morbidity and mortality, being desirable for specifically vulnerable populations, as children and elderly patients, and offering alternative therapy for those without cell sources available for allogeneic HSC transplantation, benefiting patients from ethnic minorities. This approach can be easily adopted in the clinical setting for patients planned to receive autologous HSC transplant as their standard therapy, enhancing graft-versus-cancer activity with anti-CD19 specificity. Methods: High-titer third-generation lentiviral constructs were produced carrying second-generation CD19-specific CAR co-stimulated by CD28. These vectors also co-delivered HSV-sr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC isolated from umbilical cord blood and G-CSF-mobilized apheresed peripheral blood stem cells (PBSC) were transduced with such lentiviral vectors and injected into NSG pups after irradiation for in vivo evaluation of engraftment, function and suicide gene activation. Results: We have consistently achieved engraftment of human cells in about 95% of study mice, with engraftment of CAR+ cells in about 80% of the animals. Human HSC were successfully transduced with lentiviral vectors carrying anti-CD19 CAR with no impairment of differentiation or proliferation in vitro and in vivo. Immune cells differentiated in vivo from CAR+ HSC had antigen-specific cytotoxicity directed by CAR. CAR+ human cells were detected in BM, spleen, blood and thymus of injected mice. CAR+ T cells were stably detected in the blood of engrafted mice up to 40 weeks post-injection, demonstrating lymphopoiesis of CAR+ T cells successfully escaping thymic deletion and persisting throughout murine lifetime. As a surrogate of the antigen specificity and efficacy, CD19+ cells were significantly decreased in all mice engrafted with anti-CD19 CAR demonstrating that CAR+ immune cells were not inactivated or developed tolerance. Mice humanized with at least 4% of CAR+ cells in blood had significant protection against challenge with CD19+ tumor cell line, with inhibition or elimination of tumor development and consequent survival advantage. Activation of HSV-sr39TK suicide gene by ganciclovir treatment successfully led to ablation of gene-modified cells in vitro and in vivo. Conclusions: Our results demonstrate feasibility of CAR modification of human HSC for cancer immunotherapy. It could be easily employed in the context of HSC transplantation to augment the anti-cancer activity, with CAR-expressing myeloid and NK cells to ensure tumor-specific immunity until de novo production of T cells from CAR-modified HSC. Disclosures Larson: BMS: Consultancy.

CD44 Is Selectively Required for the Homing and Engraftment of BCR-ABL-Expressing Leukemic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 743-743
Author(s):  
Daniela S. Krause ◽  
Katherine Lazarides ◽  
Ulrich H. von Andrian ◽  
Richard A. Van Etten
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Bone Marrow Niche ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract In chronic myeloid leukemia (CML) patients treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft can contribute to relapse of leukemia (Deisseroth et al., Blood1994; 83:3068), but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Although the frequency of autografting in CML has decreased following the introduction of imatinib, most imatinib-responsive patients harbor residual BCR-ABL-expressing stem cells (Graham et al., Blood2002; 99:319) and some will develop progressive leukemia. Autografting with cells harvested at the time of minimal residual disease could be an important salvage therapy, but methods to selectively block the engraftment of leukemic stem cells are needed. In this study, we show that CD44 expression is increased on murine BCR-ABL-expressing stem/progenitor cells and contributes to the ability of these cells to bind to Selectins. In a retroviral transduction/transplantation model of CML, BCR-ABL-transduced progenitors from CD44-deficient donors were defective in homing to recipient marrow, resulting in defective engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engrafted as efficiently as wild-type HSC. In addition, CD44 was not required for induction of acute B-lymphoblastic leukemia (B-ALL) by BCR-ABL, indicating that the engraftment requirement for CD44 is specific to leukemic cells initiating CML, not B-ALL. The requirement for donor CD44 was bypassed by direct intrafemoral injection of BCR-ABL-transduced CD44-deficient stem cells, or by co-expression of human CD44 with BCR-ABL. Treatment of BCR-ABL-transduced stem/progenitor cells from wild-type donors with antibody to CD44 attenuated the induction of CML-like leukemia in recipients. These results demonstrate a major role for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells, possibly through adhesive interactions with Selectins and/or hyaluronan in the recipient bone marrow niche. They further argue that BCR-ABL-transduced stem/progenitor cells depend to a greater extent on CD44 for engraftment than do normal HSC, and suggest that CD44 blockade may be beneficial in autologous transplantation in CML.

scholarly journals Hematopoietic stem cells acquire survival advantage by loss of RUNX1 methylation identified in familial leukemia

Blood ◽  
2020 ◽  
Vol 136 (17) ◽  
pp. 1919-1932
Author(s):  
Takayoshi Matsumura ◽  
Ayako Nakamura-Ishizu ◽  
Siva Sai Naga Anurag Muddineni ◽  
Darren Qiancheng Tan ◽  
Chelsia Qiuxia Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Posttranslational Modifications ◽  
Dominant Negative ◽  
Survival Advantage ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Genetic Ablation

Abstract RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress–induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

scholarly journals Druggable Biochemical Pathways and Potential Therapeutic Alternatives to Target Leukemic Stem Cells and Eliminate the Residual Disease in Chronic Myeloid Leukemia

2019 ◽  
Vol 20 (22) ◽  
pp. 5616 ◽  
Author(s):  
Fabien Muselli ◽  
Jean-François Peyron ◽  
Didier Mary
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Hematopoietic Stem

Chronic Myeloid Leukemia (CML) is a disease arising in stem cells expressing the BCR-ABL oncogenic tyrosine kinase that transforms one Hematopoietic stem/progenitor Cell into a Leukemic Stem Cell (LSC) at the origin of differentiated and proliferating leukemic cells in the bone marrow (BM). CML-LSCs are recognized as being responsible for resistances and relapses that occur despite the advent of BCR-ABL-targeting therapies with Tyrosine Kinase Inhibitors (TKIs). LSCs share a lot of functional properties with Hematopoietic Stem Cells (HSCs) although some phenotypical and functional differences have been described during the last two decades. Subverted mechanisms affecting epigenetic processes, apoptosis, autophagy and more recently metabolism and immunology in the bone marrow microenvironment (BMM) have been reported. The aim of this review is to bring together the modifications and molecular mechanisms that are known to account for TKI resistance in primary CML-LSCs and to focus on the potential solutions that can circumvent these resistances, in particular those that have been, or will be tested in clinical trials.

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