| 1. |
- Herold, Nikolas, et al.
(författare)
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Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies
- 2017
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 23:2, s. 256-263
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Tidskriftsartikel (refereegranskat)abstract
- The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults'. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP)(2-5), which causes DNA damage through perturbation of DNA synthesis(6). Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment(7-9). Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.
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| 2. |
- Heshmati, Yaser
(författare)
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Identification and characterization of novel genetic and epigenetic factors required for normal and malignant hematopoiesis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukemia (AML) is a type of blood cancer, characterized by clonal expansion and loss of differentiation ability of myeloid progenitor cells leading to abnormal accumulation of immature myeloid cells (myeloblasts) in the bone marrow and peripheral blood. This thesis (study I to IV) focused on the identification and characterization of genes which are required for AML growth. The final study (study V) aimed to uncover the role of NAP1L3 in normal hematopoietic stem cells (HSCs). In studies I and II, we performed large-scale RNA interference screens in mouse and AML human cell lines to identify novel factors and pathways required for AML growth. Using this approach, we identified two novel targets: Chromatin remodeling factor CHD4 (study I) and the transcription factor GTF2IRD1 (study II), which display both a strong inhibitory effect on the growth of AML cells and a less negative effect on normal hematopoietic cells. Using RNA interference and CRISPR-Cas9 techniques, we revealed that these genes were crucial for AML cell growth in vitro and in vivo. Knockdown of either CHD4 or GTF2IRD1 accumulated cells in the G0 phase of the cell cycle and resulted in downregulation of MYC and its target genes. We demonstrated the inhibitory role of CHD4 knockdown on the growth and maintenance of primary childhood AML in an ex vivo setting, as well as in a xenograft model by transplanting patient-derived samples into humanized NSG-SGM3 mice. GTF2IRD1 knockdown reduced the number of primary childhood and adult AML cells in ex vivo culture and delayed AML progression in the transplanted animal model. Therefore, CHD4 and GTF2IRD1 are important for AML cell growth, and interestingly the knockdown of these two genes did not show a strong inhibitory effect on normal hematopoietic cell growth. In study III, we described the role of an epigenetic enzyme, the histone methyl-transferase EHMT1 in AML. We used RNA interference, CRISPR-Cas9, and pharmacological approaches to inhibit EHMT1 expression, which prevented the growth of various AML cell lines and primary AML patient samples. Knockdown of EHMT1 significantly delayed disease progression in AML mouse models and prolonged their survival. Next, we employed CRISPR-Cas9 technology to generate single and double gene knockouts of EHMT1 and its homolog EHMT2, which showed that both enzymes cooperatively play a role in AML cell proliferation and shared a similar cellular mechanism as individual knockouts of either gene resulted in an increased number of cells in G0 phase of the cell cycle. RNA sequencing of the transcriptome of AML cells with EHMT1 and EHMT2 knockdown identified several common biological processes, including cell differentiation, proliferation and survival, as well as other unshared pathways and downstream effectors. In study IV, we contributed to Nikolas Herold’s study, who found that deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) plays a role in detoxifying intracellular ara-CTP in cells treated with the deoxycytidine analog cytarabine (ara-C). Transient reduction of SAMHD1 expression by using the simian immunodeficiency virus (SIV) protein Vpx significantly increased the sensitivity of AML cells to ara-C, whereas AML cells lacking SAMHD1 transplanted into recipient mice were hypersensitive to ara-C. We showed that in vitro treatment of primary AML patient samples with Vpx, which suppresses SAMHD1, resulted in reduced proliferation of AML but not normal cells. Together, our data suggest that SAMHD1 inhibition can be used as a therapeutic strategy for cancer (AML) patients with high SAMHD1 expression. In study V, our aim was to identify novel epigenetic regulators of normal HSCs. We found high expression level of Nap1l3, a member of nucleosome assembly proteins (NAPs), as a histone chaperone in HSCs. Loss of function of mouse Nap1l3 mediated by shRNA or CRISPR-Cas9 impaired the maintenance and differentiation of HSCs in both our in vitro and in vivo studies. Moreover, downregulation of NAP1L3 in human UCB HSCs significantly decreased both the number of colonies formed by HSCs and their proliferation in vitro due to cell cycle arrest in the G0 phase. Xenograft mouse models using human HSCs with NAP1L3 knockdown showed a reduction of HSC reconstitution and bias in differentiation. Furthermore, we observed upregulation of several HOX genes (HOXA3, HOXA5, HOXA6 and HOXA9) under NAP1L3 suppression in human HSCs. Altogether, in this thesis, we showed the important roles of CHD4, EHMT1 and GTF2IRD1 in AML cell expansion, identifying them as potential novel targets for AML treatment. Moreover, we revealed the cellular mechanisms and RNA expression patterns under knockdown of these genes. We contributed to the study that found SAMHD1 expression level can be used as a prognostic marker for ara-C treatment and that inhibition of SAMHD1 increases the sensitivity of AML cells to ara-C treatment. Finally, we identified a novel regulatory role for NAP1L3 as a histone chaperone in self-renewal and differentiation of HSCs.
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| 3. |
- Luu, Thuy T., et al.
(författare)
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FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15R beta (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.
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| 4. |
- Sanjiv, Kumar, et al.
(författare)
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MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia
- 2021
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Ingår i: Cancer Research. - : American Association For Cancer Research (AACR). - 0008-5472 .- 1538-7445. ; 81:22, s. 5733-5744
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Tidskriftsartikel (refereegranskat)abstract
- Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin−CD34+CD38−), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial.
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