The nonessential amino acid cysteine is required to prevent ferroptosis in acute myeloid leukemia.

ABSTRACT: Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing.

Dietary methionine starvation impairs acute myeloid leukemia progression.

Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance-based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

Inhibition of the succinyl dehydrogenase complex in acute myeloid leukemia leads to a lactate-fuelled respiratory metabolic vulnerability.

Metabolic programs can differ substantially across genetically distinct subtypes of acute myeloid leukemia (AML). These programs are not static entities but can change swiftly as a consequence of extracellular changes or in response to pathway-inhibiting drugs. Here, we uncover that AML patients with FLT3 internal tandem duplications (FLT3-ITD+) are characterized by a high expression of succinate-CoA ligases and high activity of mitochondrial electron transport chain (ETC) complex II, thereby driving high mitochondrial respiration activity linked to the Krebs cycle. While inhibition of ETC complex II enhances apoptosis in FLT3-ITD+ AML, cells also quickly adapt by importing lactate from the extracellular microenvironment. 13C3-labelled lactate metabolic flux analyses reveal that AML cells use lactate as a fuel for mitochondrial respiration. Inhibition of lactate transport by blocking Monocarboxylic Acid Transporter 1 (MCT1) strongly enhances sensitivity to ETC complex II inhibition in vitro as well as in vivo. Our study highlights a metabolic adaptability of cancer cells that can be exploited therapeutically.

The Glycolytic Gatekeeper PDK1 defines different metabolic states between genetically distinct subtypes of human acute myeloid leukemia.

Acute myeloid leukemia remains difficult to treat due to strong genetic heterogeneity between and within individual patients. Here, we show that Pyruvate dehydrogenase kinase 1 (PDK1) acts as a targetable determinant of different metabolic states in acute myeloid leukemia (AML). PDK1low AMLs are OXPHOS-driven, are enriched for leukemic granulocyte-monocyte progenitor (L-GMP) signatures, and are associated with FLT3-ITD and NPM1cyt mutations. PDK1high AMLs however are OXPHOSlow, wild type for FLT3 and NPM1, and are enriched for stemness signatures. Metabolic states can even differ between genetically distinct subclones within individual patients. Loss of PDK1 activity releases glycolytic cells into an OXPHOS state associated with increased ROS levels resulting in enhanced apoptosis in leukemic but not in healthy stem/progenitor cells. This coincides with an enhanced dependency on glutamine uptake and reduced proliferation in vitro and in vivo in humanized xenograft mouse models. We show that human leukemias display distinct metabolic states and adaptation mechanisms that can serve as targets for treatment.

Transcriptional regulators CITED2 and PU.1 cooperate in maintaining hematopoietic stem cells.

Reduced expression of the transcription factor PU.1 is frequently associated with development of acute myeloid leukemia (AML), whereas elevated levels of CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) enhance maintenance of both normal and leukemic hematopoietic stem and progenitor cells (HSPCs). Recent findings indicate that PU.1 and CITED2 act in the same gene regulatory network. We therefore examined a potential synergistic effect of simultaneous PU.1 downregulation and CITED2 upregulation on stem cell biology and AML pathogenesis. We found that simultaneous PU.1/CITED2 deregulation in human CD34+ cord blood (CB) cells, as well as CITED2 upregulation in preleukemic murine PU.1-knockdown (PU.1KD/KD) bone marrow cells, significantly increased the maintenance of HSPCs compared with the respective deregulation of either factor alone. Increased replating capacity of PU.1KD/KD/CITED2 cells in in vitro assays eventually resulted in outgrowth of transformed cells, while upregulation of CITED2 in PU.1KD/KD cells enhanced their engraftment in in vivo transplantation studies without affecting leukemic transformation. Transcriptional analysis of CD34+ CB cells with combined PU.1/CITED2 alterations revealed a set of differentially expressed genes that highly correlated with gene signatures found in various AML subtypes. These findings illustrate that combined PU.1/CITED2 deregulation induces a transcriptional program that promotes HSPC maintenance, which might be a prerequisite for malignant transformation.

CITED2 affects leukemic cell survival by interfering with p53 activation.

CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) is a regulator of the acetyltransferase CBP/p300 and elevated CITED2 levels are shown in a number of acute myeloid leukemia (AML). To study the in vivo role of CITED2 in AML maintenance, AML cells were transduced with a lentiviral construct for RNAi-mediated knockdown of CITED2. Mice transplanted with CITED2-knockdown AML cells (n=4) had a significantly longer survival compared to mice transplanted with control AML cells (P<0.02). In vitro, the reduction of CITED2 resulted in increased p53-mediated apoptosis and CDKN1A expression, whereas BCL2 levels were reduced. The activation of p53 upon CITED2 knockdown is not a direct consequence of increased CBP/p300-activity towards p53, since no increased formation of CBP/p300/p53 complexes was demonstrated and inhibition of CBP/p300-activity could not rescue the phenotype of CITED2-deficient cells. Instead, loss of CITED2 had an inhibitory effect on the AKT-signaling pathway, which was indicated by decreased levels of phosphorylated AKT and altered expression of the AKT-pathway regulators PHLDA3 and SOX4. Notably, simultaneous upregulation of BCL2 or downregulation of the p53-target gene PHLDA3 rescued the apoptotic phenotype in CITED2-knockdown cells. Furthermore, knockdown of CITED2 led to a decreased interaction of p53 with its inhibitor MDM2, which results in increased amounts of total p53 protein. In summary, our data indicate that CITED2 functions in pathways regulating p53 activity and therefore represents an interesting target for AML therapy, since de novo AML cases are characterized by an inactivation of the p53 pathway or deregulation of apoptosis-related genes.

Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis.

Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34(+) cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

Nonredundant and locus-specific gene repression functions of PRC1 paralog family members in human hematopoietic stem/progenitor cells.

The Polycomb group (PcG) protein BMI1 is a key factor in regulating hematopoietic stem cell (HSC) and leukemic stem cell self-renewal and functions in the context of the Polycomb repressive complex 1 (PRC1). In humans, each of the 5 subunits of PRC1 has paralog family members of which many reside in PRC1 complexes, likely in a mutually exclusive manner, pointing toward a previously unanticipated complexity of Polycomb-mediated silencing. We used an RNA interference screening approach to test the functionality of these paralogs in human hematopoiesis. Our data demonstrate a lack of redundancy between various paralog family members, suggestive of functional diversification between PcG proteins. By using an in vivo biotinylation tagging approach followed by liquid chromatography-tandem mass spectrometry to identify PcG interaction partners, we confirmed the existence of multiple specific PRC1 complexes. We find that CBX2 is a nonredundant CBX paralog vital for HSC and progenitor function that directly regulates the expression of the cyclin-dependent kinase inhibitor p21, independently of BMI1 that dominantly controls expression of the INK4A/ARF locus. Taken together, our data show that different PRC1 paralog family members have nonredundant and locus-specific gene regulatory activities that are essential for human hematopoiesis.

Single-cell STAT5 signal transduction profiling in normal and leukemic stem and progenitor cell populations reveals highly distinct cytokine responses.

BACKGROUND: Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. However, the manner in which STAT5 responds to early-acting and lineage-restricted cytokines, particularly in leukemic stem/progenitor cells, is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at a single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34(+)CD38(-) hematopoietic stem cells (HSCs). EPO- and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34(+) cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34(+) cells on stroma. CONCLUSIONS/SIGNIFICANCE: In conclusion, our optimized multiparametric flow cytometry protocols allow the analysis of signal transduction at the single cell level in normal and leukemic stem and progenitor cells. Our study demonstrates highly distinctive cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.

Expansion of normal and leukemic human hematopoietic stem/progenitor cells requires rac-mediated interaction with stromal cells.

OBJECTIVE: To determine the involvement of Rac signaling in self-renewal and expansion on bone marrow stroma of normal CD34+ cells vs leukemic CD34+ cells from acute myeloid leukemia (AML) patients. MATERIALS AND METHODS: Rac signaling was modulated by retroviral introduction of Racl-N17, Racl-V12, or by using the Rac inhibitor NSC23766. In long-term MS5 cocultures (leukemic) expansion, migration, adhesion, and presence of stem/progenitor cells were monitored in both normal as well as leukemic CD34+ cells. RESULTS: Inhibition of Rac signaling impaired migration and adhesion of cord blood (CB) CD34+ cells on MS5 stroma. Long-term inhibition of Rac during a 5-week coculture period on stroma prevented association of hematopoietic progenitors with the bone marrow stromal cells and resulted in a dramatic decrease in the primitive stem cell frequency (long-term culture-initiating cell) in a dose-dependent manner. Many of these phenotypes were reversed in the presence of activated Racl-V12, including improved migration toward, and association with, MS5 cells. CD34+ AML cells were characterized by elevated levels of Rac activity (five of seven patients) and enhanced migration and adhesion to MS5 bone marrow stroma as compared to CB CD34+ cells. A dramatic decrease was observed in the formation of leukemic cobblestone area-forming cells as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. CONCLUSION: Our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.

HSP27 protects AML cells against VP-16-induced apoptosis through modulation of p38 and c-Jun.

OBJECTIVE: To investigate 1) the signal transduction pathways affected by heat shock protein 27 (HSP27) expression; and 2) the expression and regulation of HSP27 in acute myeloid leukemia (AML). MATERIALS AND METHODS: RNA interference studies for HSP27 in leukemic TF-1 cells were used to investigate the effects on downstream signal transduction and apoptosis after VP-16 and CD95/Fas treatment. HSP27 expression and activation was investigated in AML blasts through Western blot analysis. RESULTS: RNA interference for HSP27 resulted in a twofold increase in VP-16-induced apoptosis, which was preceded by enhanced p38 and c-Jun phosphorylation and a twofold increased cytochrome c release into the cytoplasm. DAXX co-immunoprecipitated with HSP27, suggesting an inhibitory role of HSP27 in VP-16-mediated activation of the ASK1/p38/JNK pathway. CD95/Fas-induced apoptosis, however, was unaffected by HSP27 siRNA, due to upregulation of HSP27. Although HSP27 was highly expressed and phosphorylated in primitive monocytic AML blasts (M4-M5, 91%, n=11) and undetectable in myeloid blasts (M1-M2, n=5), VP-16-mediated apoptosis correlated moderately with HSP27 expression. This is likely due to the co-expression of p21Waf1/Cip1, which is in the majority of the monocytic AML M4-M5 blasts constitutively localized in the cytoplasm. Overexpression of cytoplasmic p21 inhibited the enhanced p38 phosphorylation after HSP27 RNAi, suggesting a predominant anti-apoptotic role of p21 over HSP27. CONCLUSION: 1) HSP27 inhibits VP-16-mediated phosphorylation of p38 and c-Jun, cytochrome c release, and subsequent apoptosis; 2) HSP27 is expressed and activated in monocytic AML blasts; 3) cytoplasmic expression of p21 compensates for the lack of HSP27.

Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene.

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.