Aims: Adaptation to low oxygen of hematopoietic stem cells (HSCs) in the bone marrow has been demonstrated to depend on the activation of hypoxia-inducible factor (HIF)-1α as well as the limited production of reactive oxygen species (ROS). In this study, we aimed at determining whether HIF-1α is involved in protecting HSCs from ROS.

Results: Oxidative stress was induced by DL-buthionine-(S,R)-sulfoximine (BSO)-treatment, which increases the mitochondrial ROS level. Hypoxia rescued Lineage-Sca-1⁺c-kit⁺ (LSK) cells from BSO-induced apoptosis, whereas cells succumbed to apoptosis in normoxia. Apoptosis in normoxia was inhibited with the antioxidant N-acetyl-L-cysteine or by overexpression of anti-apoptotic BCL-2. Moreover, stabilized expression of oxygen-insensitive HIFs could not protect LSK cells from oxidative stress-induced apoptosis at normoxia, neither could short hairpin RNA to Hif-1α inhibit the protective effects by hypoxia in LSK cells. Likewise, BSO treatment of LSK cells from Hif-1α knockout mice did not suppress the effects seen in hypoxia. Microarray analysis identified the nuclear factor-kappa B (NF-κB) pathway as a pathway induced by hypoxia. By using NF-κB lentiviral construct and DNA-binding assay, we found increased NF-κB activity in cells cultured in hypoxia compared with normoxia. Using an inhibitor against NF-κB activation, we could confirm the involvement of NF-κB signaling as BSO-mediated cell death was significantly increased in hypoxia after adding the inhibitor.

Innovation: HIF-1α is not involved in protecting HSCs and progenitors to elevated levels of ROS on glutathione depletion during hypoxic conditions.

Conclusion: The study proposes a putative role of NF-κB signaling as a hypoxia-induced regulator in early hematopoietic cells.

This thesis has studied the role of low oxygen levels, or hypoxia, in hematopoietic stem cells (HSCs) and how, at the molecular level, it regulates stem cell maintenance and protects against oxidative stress induced by reactive oxygen species (ROS). HSCs reside within the bone marrow in specific niches created by a unique vascularized environment, which is suggested to be hypoxic and crucial for HSCs by maintaining a quiescent state of cell cycle and by redirecting metabolism away from the mitochondria to glycolysis. The niches are also believed to limit the production of ROS, which could damage DNA and disrupt the stem cell features. The hypoxia-responsive protein hypoxia-inducible factor 1 alpha (HIF-1α) is a major regulator of the hypoxic cell response in HSCs as well as in leukemic stem cells. Both these cells are thought to reside in the bone marrow where they are protected from stress and chemotherapy by niche cells and hypoxia.

The thesis demonstrates that pyruvate dehydrogenase kinase 1 regulates a metabolic shift to glycolysis, and maintains the engraftment potential of both HSCs and multipotent progenitors upon transplantation. Furthermore, we wanted to determine whether HIF-1α or other signaling pathways are involved in protecting HSCs from ROS-induced cell death. Overexpression, silencing or a knockout mouse model of Hif-1α could not identify HIF-1α as important for protecting HSCs from oxidative stress-induced cell death through inhibition of synthesis of the antioxidant glutathione. Gene expression analysis instead identified the transcription factor nuclear factor kappa B (NF-κB) as induced by hypoxia. By studying NF- κB signaling we found increased NF-κB activity in cells cultured in hypoxia compared to normoxia. Suppression of inhibitor of kappa B indicated a putative role of NF-κB signaling in hypoxia-induced protection against oxidative stress. The findings show that hypoxia-induced protection to elevated levels of ROS upon glutathione depletion seems to be attributed to activation of the NF-κB signaling pathway independently of HIF-1α.

To address the question whether hypoxic in vitro cultures support maintenance and promote HSC expansion we performed a limited dilution-transplantation assay. Our data indicate that hypoxic cultures maintain more long-term-reconstituting HSCs than normoxia, but this could not be confirmed statistically. Finally, we wanted to study the mechanisms by which hypoxia protect against chemotherapy. We could demonstrate that hypoxic culture protects leukemic cell lines against apoptosis induced by chemotherapy or inhibitors used for treatment of leukemia. This multidrug resistance seems to be mediated by ATP-binding cassette transporter genes, which are upregulated by hypoxia and whose inhibition has been shown to increase chemosensitivity. In addition, HIF-1α was upregulated in the leukemic cell lines in hypoxia and its inhibition increased the sensitivity to chemotherapy, indicating a role in inducing chemotherapy resistance.

Conclusively, the results presented in this thesis stress the importance of hypoxia in regulating metabolism, oxidative-stress response and maintenance of both HSCs as well as leukemic cells, especially through the critical transcription factors HIF-1α and NF-κB and their target genes.  

The phosphatidylinositol 3-kinase/AKT pathway is an integral component of signaling involved in the development of many cancers, including myeloid leukemias such as chronic myeloid leukemia and acute myeloid leukemia (AML). Increased AKT1 activity is frequently seen in AML patients, providing leukemic cells with growth and survival promoting signals. An important aspect of AKT1 function is its involvement in cellular metabolism and energy production. Under some circumstances, strong activation of AKT1 increases oxidative stress, which can cause apoptosis when cells progressively build up excess free radicals. This has been described in hematopoietic cells overexpressing activated AKT1; however, whether this is true in cells coexpressing other genetic events involved in leukemia is not known. This prompted us to investigate the effect of constitutively active AKT1 (myristoylated AKT1) in hematopoietic progenitor cells expressing constitutively active signal transducer and activator of transcription 5, Fms-related tyrosine kinase 3-internal tandem duplication, or antiapoptotic B-cell lymphoma 2. Surprisingly, myristoylated AKT1 was incompatible with proliferation driven by both signal transducer and activator of transcription 5 and Fms-related tyrosine kinase 3-internal tandem duplication, which triggered cell cycle block and apoptosis. Moreover, transplantable cells of B-cell lymphoma 2-transgenic mice were impaired in their engraftment ability to recipient mice when expressing hyperactivated AKT1. This Was linked to AKT1-mediated proapoptotic functions and not to impairment in homing to the bone marrow. Although cells expressing hyperactivated AKT1 displayed higher levels of reactive oxygen species both in vitro and in vivo, the addition of the antioxidant N-acetyl-L-cysteine significantly reduced apoptosis. Taken together, the results indicate that constitutive AKT1 activity is incompatible with growth- and survival-promoting ability of other activated genes in AML. Copyright (C) 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.

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Internal tandem duplications (ITD) in the FMS like tyrosine kinase (FLT3) receptor are one of the most common classes of mutations in acute myeloid leukemia (AML), which presence indicates a poor prognosis. Lengths of FLT3-ITD mutations found in patients can vary from 3 up to hundreds of nucleotides and may be located either in the juxtamembrane domain or the tyrosine kinase-1 domain (TKD1). There are contradicting opinions whether the length of the ITD has an impact on the clinical situation and whether tyrosines duplicated are of any significance for oncogenic signaling. Considering the substantial differences in lengths as well as the variability of start and end points of ITDs, we have performed a study of various FLT3-ITD mutations isolated from AML-patients. The ITD region from leukemic blasts of nine AML patients were sequenced and cloned by PCR into the human wildtype FLT3 cDNA, inserted to a retroviral GFP-containing vector. The hematopoietic progenitor cell line FDC-P1 was used to elucidate the impact of the different ITDs on growth, survival, signal transduction, and resistance to the FLT3-targeting inhibitor PKC412. Interestingly, the shortest and the longest ITDs were two of the three mutations that lead to the poorest survival of cells upon cytokine-deprivation, indicating that ITD size may not influence the transforming potential of cells. Furthermore one ITD that starts and ends relatively 3´ positioned, and comprises the 5´-part of the TKD1 showed both a survival advantage in starvation experiments and a significantly higher proliferation potential in comparison to several other mutations. Two other ITDs spanning this region, but with more 5´localized starting points, displayed less sensitivity to PKC412 treatment. However, this was not associated to STAT5 activity and MCL-1 upregulation as suggested by previous report. Taken together, this study suggests that different FLT3-ITD mutations may induce distinct signaling and response towards FLT3 targeting drugs, dependent of FLT3-ITD composition and not length.

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Increased AKT activity is frequently seen in patients with acute myeloid leukemia (AML), providing leukemic cells with both growth-promoting and survival signals involved in the transformation process. In AML up to 30% of all patients carry activating mutations in the tyrosine kinase receptor FLT3, leading to activation of the PI3K/AKT pathway as well as STAT5. Here, we investigated the effect of hyperactivated AKT (myristylated AKT) by retroviral transfer to hematopoietic progenitor cells coexpressing STAT5, FLT3-ITD, or antiapoptotic Bcl-2. AKT was unable to relieve cytokine-dependence. Surprisingly, uncontrolled AKT activity was linked to accumulation of cells in the G0 stage of the cell cycle and increased cell numbers became apoptotic. Hyperactivated AKT was incompatible with STAT5-driven proliferation and triggered apoptosis. The same was true also in FLT3-ITDexpressing progenitor cells of transgenic mice. Transplantable hematopoietic stem cells of wildtype and Bcl-2 transgenic mice were impaired in their engraftment ability to recipient mice when expressing hyperactivated AKT. This was linked to AKT-mediated pro-apoptotic functions and not due to effects on homing or migration. Cells expressing hyperactivated AKT displayed higher levels of reactive oxygen species. However, the addition of the antioxidant N-acetyl-L-lysine significantly reduced apoptosis. Taken together, the results indicate that constitutive AKT activity is incompatible with the growth- and survivalpromoting ability of FLT3-ITD and its downstream targets. These findings may provide a novel tool to intervene with AKT activity in leukemia.