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This file describes all yeast experiments and includes supplementary tables S1-S3 and supplementary figures S1-S10.

High expression of VEGFC predicts adverse prognosis in acute myeloid leukemia (AML). We therefore explored VEGFC-targeting efficacy as an AML therapy using a VEGFC mAb. VEGFC antibody therapy enforced myelocytic differentiation of clonal CD34+ AML blasts. Treatment of CD34+ AML blasts with the antibody reduced expansion potential by 30% to 50% and enhanced differentiation via FOXO3A suppression and inhibition of MAPK/ERK proliferative signals. VEGFC antibody therapy also accelerated leukemia cell differentiation in a systemic humanized AML mouse model. Collectively, these results define a regulatory function of VEGFC in CD34+ AML cell fate decisions via FOXO3A and serve as a new potential differentiation therapy for patients with AML.Significance: These findings reveal VEGFC targeting as a promising new differentiation therapy in AML. Cancer Res; 78(20); 5940–8. ©2018 AACR.

Suppl. figure 2: PDX samples have different basal NR3C1 expression levels.

ChIP-seq data for NR3C1 bound genes

Purpose:KPT-8602 (Eltanexor) is a second-generation exportin-1 (XPO1) inhibitor with potent activity against acute lymphoblastic leukemia (ALL) in preclinical models and with minimal effects on normal cells. In this study, we evaluated whether KPT-8602 would synergize with dexamethasone, vincristine, or doxorubicin, three drugs currently used for the treatment of ALL.Experimental Design:First, we searched for the most synergistic combination of KPT-8602 with dexamethasone, vincristine, or doxorubicin in vitro in both B-ALL and T-ALL cell lines using proliferation and apoptosis as a readout. Next, we validated this synergistic effect by treatment of clinically relevant B- and T-ALL patient-derived xenograft models in vivo. Finally, we performed RNA-sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the mechanism of synergy.Results:KPT-8602 showed strong synergism with dexamethasone on human B-ALL and T-ALL cell lines as well as in vivo in three patient-derived ALL xenografts. Compared with single-drug treatment, the drug combination caused increased apoptosis and led to histone depletion. Mechanistically, integration of ChIP-seq and RNA-seq data revealed that addition of KPT-8602 to dexamethasone enhanced the activity of the glucocorticoid receptor (NR3C1) and led to increased inhibition of E2F-mediated transcription. We observed strong inhibition of E2F target genes related to cell cycle, DNA replication, and transcriptional regulation.Conclusions:Our preclinical study demonstrates that KPT-8602 enhances the effects of dexamethasone to inhibit B-ALL and T-ALL cells via NR3C1- and E2F-mediated transcriptional complexes, allowing to achieve increased dexamethasone effects for patients.

Complete supplementary Data file including supportive figures that highlight: VEGFC targeting potential in pediatric AML, VEGFC/KDR complex molecular targeting in AML, and VEGFC antibody induced differentiation phenotype in AML, and supplementary tables include the patient characteristics, used antibodies and primer sequences, and FLT3-ITD ratio's

RNA-seq data

This file contains 5 supplementary figures containing key data that could not be fitted in the main figures.

Suppl. figure 1: Doxorubicin and vincristine are not synergistic with KPT-8602 in diminishing proliferation of B-ALL and T-ALL

Suppl. figure 4: KPT-8602 single treatment or combined treatment with dexamethasone does not lead to nuclear accumulation of IKBα nor to effects on NFκB target genes

Ribosomopathies are congenital disorders caused by mutations in ribosomal proteins (RP) or assembly factors and are characterized by cellular hypoproliferation at an early stage. Paradoxically, many of these disorders have an elevated risk to progress to hyperproliferative cancer at a later stage. In addition, somatic RP mutations have recently been identified in various cancer types, for example, the recurrent RPL10-R98S mutation in T-cell acute lymphoblastic leukemia (T-ALL) and RPS15 mutations in chronic lymphocytic leukemia (CLL). We previously showed that RPL10-R98S promotes expression of oncogenes, but also induces a proliferative defect due to elevated oxidative stress. In this study, we demonstrate that this proliferation defect is eventually rescued by RPL10-R98S mouse lymphoid cells that acquire 5-fold more secondary mutations than RPL10-WT cells. The presence of RPL10-R98S and other RP mutations also correlated with a higher mutational load in patients with T-ALL, with an enrichment in NOTCH1-activating lesions. RPL10-R98S–associated cellular oxidative stress promoted DNA damage and impaired cell growth. Expression of NOTCH1 eliminated these phenotypes in RPL10-R98S cells, in part via downregulation of PKC-θ, with no effect on RPL10-WT cells. Patients with RP-mutant CLL also demonstrated a higher mutational burden, enriched for mutations that may diminish oxidative stress. We propose that oxidative stress due to ribosome dysfunction causes hypoproliferation and cellular insufficiency in ribosomopathies and RP-mutant cancer. This drives surviving cells, potentiated by genomic instability, to acquire rescuing mutations, which ultimately promote transition to hyperproliferation.Significance:Ribosomal lesions cause oxidative stress and increase mutagenesis, promoting acquisition of rescuing mutations that stimulate proliferation.

Suppl. figure 3: Dexamethasone and dexamethasone+KPT-8602 treatment induce NR3C1 binding and leads to differential gene expression of the bound genes in the SUP-T1 cell line

Background One-third of cancers activate endogenous synthesis of serine/glycine, and can become addicted to this pathway to sustain proliferation and survival. Mechanisms driving this metabolic rewiring remain largely unknown. Methods NKX2–1 overexpressing and NKX2–1 knockdown/knockout T-cell leukaemia and lung cancer cell line models were established to study metabolic rewiring using ChIP-qPCR, immunoblotting, mass spectrometry, and proliferation and invasion assays. Findings and therapeutic relevance were validated in mouse models and confirmed in patient datasets. Results Exploring T-cell leukaemia, lung cancer and neuroendocrine prostate cancer patient datasets highlighted the transcription factor NKX2–1 as putative driver of serine/glycine metabolism. We demonstrate that transcription factor NKX2–1 binds and transcriptionally upregulates serine/glycine synthesis enzyme genes, enabling NKX2–1 expressing cells to proliferate and invade in serine/glycine-depleted conditions. NKX2–1 driven serine/glycine synthesis generates nucleotides and redox molecules, and is associated with an altered cellular lipidome and methylome. Accordingly, NKX2–1 tumour-bearing mice display enhanced tumour aggressiveness associated with systemic metabolic rewiring. Therapeutically, NKX2–1-expressing cancer cells are more sensitive to serine/glycine conversion inhibition by repurposed anti-depressant sertraline, and to etoposide chemotherapy. Conclusion Collectively, we identify NKX2–1 as a novel transcriptional regulator of serine/glycine synthesis addiction across cancers, revealing a therapeutic vulnerability of NKX2–1-driven cancers.

Cancer cells reprogramme their metabolism to support unrestrained proliferation and survival in nutrient-poor conditions. Whereas non-transformed cells often have lower demands for serine and glycine, several cancer subtypes hyperactivate intracellular serine and glycine synthesis and become addicted to de novo production. Copy-number amplifications of serine- and glycine-synthesis genes and genetic alterations in common oncogenes and tumour-suppressor genes enhance serine and glycine synthesis, resulting in high production and secretion of these oncogenesis-supportive metabolites. In this Review, we discuss the contribution of serine and glycine synthesis to cancer progression. By relying on de novo synthesis pathways, cancer cells are able to enhance macromolecule synthesis, neutralize high levels of oxidative stress and regulate methylation and tRNA formylation. Furthermore, we discuss the immunosuppressive potential of serine and glycine, and the essentiality of both amino acids to promoting survival of non-transformed neighbouring cells. Finally, we point to the emerging data proposing moonlighting functions of serine- and glycine-synthesis enzymes and examine promising small molecules targeting serine and glycine synthesis. ispartof: NATURE METABOLISM vol:3 issue:2 pages:131-141 ispartof: location:Germany status: published

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival, and chemotherapy resistance. Although normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis-addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking, enzymatic assays, and drug-target interaction studies revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline's antiproliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell-cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis-addicted cancers. ispartof: MOLECULAR CANCER THERAPEUTICS vol:20 issue:1 pages:50-63 ispartof: location:United States status: published

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis. ispartof: Blood vol:139 issue:21 pages:3111-3126 ispartof: location:United States status: Published online

Cancer arises when normal cells are transformed into malignant cells by acquiring a number of hallmarks such as sustained proliferative signaling; evading cell death, growth suppression and immune destruction; replicative immortality; and activation of invasion and metastasis (Hanahan et al. 2000, 2011). Sequential accumulation of genetic mutations is a major cause of acquiring these cancer hallmarks in the cell transformation process, and hence a complete characterization of the landscape of pathogenic somatic and congenital mutations in cancer cells forms a holy grail to fully understand cancer biology. Indeed, a lot of effort has gone towards characterizing somatic missense and nonsense single nucleotide variants in the protein coding regions of the genome that result in amino acid substitutions, small insertions and deletions, or a premature STOP codon in the encoded protein. Synonymous mutations on the other hand, nucleotide changes that do not result in an amino acid change in the protein for which they encode, have previously attracted significantly less at attention as candidate cancer driver mutations. However, in a variety of other diseases such as cystic fibrosis, ataxia telangiectasia and even in hereditary cancer syndromes, a causative role for synonymous mutations in disease pathogenesis has been described (Sauna et al. 2011). In addition, the number of synonymous mutations that have a significant impact on the corresponding RNA and protein expression level or isoform in different cancer types is rapidly rising. It is thus becoming clear that there might be a significant fraction of synonymous mutations that are not as ‘silent’ as they have long been considered to be. In this chapter, we will discuss why synonymous mutations have received little attention in the context of cancer. Furthermore, we will describe the recent progress that was made in characterizing the landscape of oncogenic synonymous mutations as well as the variety of molecular mechanisms by which synonymous mutations affect RNA and protein expression levels of oncogenes and tumor suppressors.

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH., The ribosomal protein RPL10 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). Here, the authors show that it promotes proliferation of T-ALL cells by upregulating the serine biosynthesis enzyme phosphoserine phosphatase which in turn modulates serine and glycine metabolism.

The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL.

Background T cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype that comprises 10–15% of childhood and 20–25% of adult ALL cases. Over 70% of T-ALL patients harbor activating mutations in the NOTCH1 signaling pathway and are predicted to be sensitive to gamma-secretase inhibitors. We have recently demonstrated that selective inhibition of PSEN1-containing gamma-secretase complexes can overcome the dose-limiting toxicity associated with broad gamma-secretase inhibitors. In this study, we developed combination treatment strategies with the PSEN1-selective gamma-secretase inhibitor MRK-560 and other targeted agents (kinase inhibitors ruxolitinib and imatinib; XPO-1 inhibitor KPT-8602/eltanexor) for the treatment of T-ALL. Methods We treated T-ALL cell lines in vitro and T-ALL patient-derived xenograft (PDX) models in vivo with MRK-560 alone or in combination with other targeted inhibitors (ruxolitinib, imatinib or KPT-8602/eltanexor). We determined effects on proliferation of the cell lines and leukemia development and survival in the PDX models. Results All NOTCH1-signaling-dependent T-ALL cell lines were sensitive to MRK-560 and its combination with ruxolitinib or imatinib in JAK1- or ABL1-dependent cell lines synergistically inhibited leukemia proliferation. We also observed strong synergy between MRK-560 and KPT-8602 (eltanexor) in all NOTCH1-dependent T-ALL cell lines. Such synergy was also observed in vivo in a variety of T-ALL PDX models with NOTCH1 or FBXW7 mutations. Combination treatment significantly reduced leukemic infiltration in vivo and resulted in a survival benefit when compared to single treatment groups. We did not observe weight loss or goblet cell hyperplasia in single drug or combination treated mice when compared to control. Conclusions These data demonstrate that the antileukemic effect of PSEN1-selective gamma-secretase inhibition can be synergistically enhanced by the addition of other targeted inhibitors. The combination of MRK-560 with KPT-8602 is a highly effective treatment combination, which circumvents the need for the identification of additional mutations and provides a clear survival benefit in vivo. These promising preclinical data warrant further development of combination treatment strategies for T-ALL based on PSEN1-selective gamma-secretase inhibition.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia that is most frequent in children and is characterized by the presence of few chromosomal rearrangements and 10 to 20 somatic mutations in protein-coding regions at diagnosis. The majority of T-ALL cases harbor activating mutations in NOTCH1 together with mutations in genes implicated in kinase signaling, transcriptional regulation, or protein translation. To obtain more insight in the level of clonal heterogeneity at diagnosis and during treatment, we used single-cell targeted DNA sequencing with the Tapestri platform. We designed a custom ALL panel and obtained accurate single-nucleotide variant and small insertion-deletion mutation calling for 305 amplicons covering 110 genes in about 4400 cells per sample and time point. A total of 108 188 cells were analyzed for 25 samples of 8 T-ALL patients. We typically observed a major clone at diagnosis (>35% of the cells) accompanied by several minor clones of which some were less than 1% of the total number of cells. Four patients had >2 NOTCH1 mutations, some of which present in minor clones, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples, we detected the presence and clonal nature of residual leukemic cells and clones with a minor presence at diagnosis that evolved to clinically relevant major clones at later disease stages. Therefore, single-cell DNA amplicon sequencing is a sensitive assay to detect clonal architecture and evolution in T-ALL. ispartof: BLOOD vol:137 issue:6 pages:801-811 ispartof: location:United States status: published

Acute leukemias (ALs) of ambiguous lineage are a heterogeneous group of high-risk leukemias characterized by coexpression of myeloid and lymphoid markers. In this study, we identified a distinct subgroup of immature acute leukemias characterized by a broadly variable phenotype, covering acute myeloid leukemia (AML, M0 or M1), T/myeloid mixed-phenotype acute leukemia (T/M MPAL), and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Rearrangements at 14q32/BCL11B are the cytogenetic hallmark of this entity. In our screening of 915 hematological malignancies, there were 202 AML and 333 T-cell acute lymphoblastic leukemias (T-ALL: 58, ETP; 178, non-ETP; 8, T/M MPAL; 89, not otherwise specified). We identified 20 cases of immature leukemias (4% of AML and 3.6% of T-ALL), harboring 4 types of 14q32/BCL11B translocations: t(2,14)(q22.3;q32) (n = 7), t(6;14)(q25.3;q32) (n = 9), t(7;14)(q21.2;q32) (n = 2), and t(8;14)(q24.2;q32) (n = 2). The t(2;14) produced a ZEB2-BCL11B fusion transcript, whereas the other 3 rearrangements displaced transcriptionally active enhancer sequences close to BCL11B without producing fusion genes. All translocations resulted in the activation of BCL11B, a regulator of T-cell differentiation associated with transcriptional corepressor complexes in mammalian cells. The expression of BCL11B behaved as a disease biomarker that was present at diagnosis, but not in remission. Deregulation of BCL11B co-occurred with variants at FLT3 and at epigenetic modulators, most frequently the DNMT3A, TET2, and/or WT1 genes. Transcriptome analysis identified a specific expression signature, with significant downregulation of BCL11B targets, and clearly separating BCL11B AL from AML, T-ALL, and ETP-ALL. Remarkably, an ex vivo drug-sensitivity profile identified a panel of compounds with effective antileukemic activity.

BACKGROUND: Thyroid carcinoma is one of the most common cancers in the world. Although the genetics of thyroid carcinoma was intensively studied, new mechanisms could be involved in its development as the codon bias. In this paper, we studied the codon bias of thyroid-cancer genes, considering not only the sequences but also the synonymous mutations. METHODS: Different measures and statistical analyses were employed to characterize the thyroid-cancer genes. We considered classical measures as RSCU and ENC, the compositional and protein characteristics, but also the codon bias landscape via the %MinMax algorithm. RESULTS: The compositional analyses highlighted two groups of thyroid cancer genes according to the GC% and GC3% content. The ENC did not show a clear codon bias in the genes. Differently, the RSCU analyses showed interesting codons that could play an important role in the development of thyroid cancer as the codon Ser-tcG. Furthermore, interesting synonymous mutations were detected that could affect the codon bias. The codon bias landscape detected genes enriched in rare codons as AKAP9 and KTN1. A cluster analysis based on %MinMax classified the thyroid cancer genes in four different groups according to the distribution of rare/frequent codons in the sequence. CONCLUSIONS: This is the first study that analyzed the codon bias in thyroid cancer genes based also on synonymous mutations. This study provided different hints that should be further investigated by wet-lab validation and that it could open new scenarios in the understanding the molecular mechanisms involved in thyroid cancer development based on codon bias. ispartof: MINERVA ENDOCRINOLOGICA vol:45 issue:4 pages:295-305 ispartof: location:Italy status: published

Metabolic rewiring is a hallmark of cancer that supports tumor growth, survival and chemotherapy resistance. While normal cells often rely on extracellular serine and glycine supply, a significant subset of cancers becomes addicted to intracellular serine/glycine synthesis, offering an attractive drug target. Previously developed inhibitors of serine/glycine synthesis enzymes did not reach clinical trials due to unfavorable pharmacokinetic profiles, implying that further efforts to identify clinically applicable drugs targeting this pathway are required. In this study, we aimed to develop therapies that can rapidly enter the clinical practice by focusing on drug repurposing, as their safety and cost-effectiveness have been optimized before. Using a yeast model system, we repurposed two compounds, sertraline and thimerosal, for their selective toxicity against serine/glycine synthesis addicted breast cancer and T-cell acute lymphoblastic leukemia cell lines. Isotope tracer metabolomics, computational docking studies and an enzymatic activity assay revealed that sertraline and thimerosal inhibit serine/glycine synthesis enzymes serine hydroxymethyltransferase and phosphoglycerate dehydrogenase, respectively. In addition, we demonstrated that sertraline’s anti-proliferative activity was further aggravated by mitochondrial inhibitors, such as the antimalarial artemether, by causing G1-S cell cycle arrest. Most notably, this combination also resulted in serine-selective antitumor activity in breast cancer mouse xenografts. Collectively, this study provides molecular insights into the repurposed mode-of-action of the antidepressant sertraline and allows to delineate a hitherto unidentified group of cancers being particularly sensitive to treatment with sertraline. Furthermore, we highlight the simultaneous inhibition of serine/glycine synthesis and mitochondrial metabolism as a novel treatment strategy for serine/glycine synthesis addicted cancers.

Purpose: KPT-8602 (Eltanexor) is a second-generation exportin-1 (XPO1) inhibitor with potent activity against acute lymphoblastic leukemia (ALL) in preclinical models and with minimal effects on normal cells. In this study, we evaluated whether KPT-8602 would synergize with dexamethasone, vincristine, or doxorubicin, three drugs currently used for the treatment of ALL. Experimental Design: First, we searched for the most synergistic combination of KPT-8602 with dexamethasone, vincristine, or doxorubicin in vitro in both B-ALL and T-ALL cell lines using proliferation and apoptosis as a readout. Next, we validated this synergistic effect by treatment of clinically relevant B- and T-ALL patient-derived xenograft models in vivo. Finally, we performed RNA-sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the mechanism of synergy. Results: KPT-8602 showed strong synergism with dexamethasone on human B-ALL and T-ALL cell lines as well as in vivo in three patient-derived ALL xenografts. Compared with single-drug treatment, the drug combination caused increased apoptosis and led to histone depletion. Mechanistically, integration of ChIP-seq and RNA-seq data revealed that addition of KPT-8602 to dexamethasone enhanced the activity of the glucocorticoid receptor (NR3C1) and led to increased inhibition of E2F-mediated transcription. We observed strong inhibition of E2F target genes related to cell cycle, DNA replication, and transcriptional regulation. Conclusions: Our preclinical study demonstrates that KPT-8602 enhances the effects of dexamethasone to inhibit B-ALL and T-ALL cells via NR3C1- and E2F-mediated transcriptional complexes, allowing to achieve increased dexamethasone effects for patients.

Ribosomopathies are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly. Intriguingly, congenital ribosomopathies display a paradoxical transition from early symptoms due to cellular hypo-proliferation to an elevated cancer risk later in life. Another association between ribosome defects and cancer came into view after the recent discovery of somatic mutations in ribosomal proteins and rDNA copy number changes in a variety of tumor types, giving rise to somatic ribosomopathies. Despite these clear connections between ribosome defects and cancer, the molecular mechanisms by which defects in this essential cellular machinery are oncogenic only start to emerge. In this review, the impact of ribosomal defects on the cellular function and their mechanisms of promoting oncogenesis are described. In particular, we discuss the emerging hallmarks of ribosomopathies such as the appearance of 'onco-ribosomes' that are specialized in translating oncoproteins, dysregulation of translation-independent extra-ribosomal functions of ribosomal proteins, rewired cellular protein and energy metabolism, and extensive oxidative stress leading to DNA damage. We end by integrating these findings in a model that can provide an explanation how ribosomopathies could lead to the transition from hypo- to hyper-proliferation in bone marrow failure syndromes with elevated cancer risk. ispartof: NUCLEIC ACIDS RESEARCH vol:48 issue:3 pages:1013-1028 ispartof: location:England status: published

Summary The anti-leukaemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphates (dNTPs), protected cells against the effects of dNTP imbalances. SAMHD1-deficient cells induced intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine acted synergistically to kill cells lacking SAMHD1. Using mass cytometry, we found that these compounds killed SAMHD1-deficient malignant cells from patients with chronic lymphocytic leukaemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation were unaffected. We therefore propose to use forodesine as a precision medicine for leukaemia, stratifying patients by SAMHD1 genotype or expression. Highlights SAMHD1-deficient cells die upon exposure to deoxyribonucleosides (dNs) Deoxyguanosine (dG) is the most toxic dN, inducing apoptosis in cells lacking SAMHD1 SAMHD1-mutated leukaemic cells can be killed by dG and the PNP-inhibitor forodesine In Brief SAMHD1 degrades deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Davenne et al. found that SAMHD1 protects cells against dNTP imbalances. Exposure of SAMHD1-deficient cells to deoxyguanosine (dG) results in increased intracellular dGTP levels and subsequent apoptosis. This can be exploited to selectively kill cancer cells that acquired SAMHD1 mutations.

The R98S mutation in ribosomal protein L10 (RPL10 R98S) affects 8% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) cases, and was previously described to impair cellular proliferation. The current study reveals that RPL10 R98S cells accumulate reactive oxygen species which promotes mitochondrial dysfunction and reduced ATP levels, causing the proliferation defect. RPL10 R98S mutant leukemia cells can survive high oxidative stress levels via a specific increase of IRES-mediated translation of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2), mediating BCL-2 protein overexpression. RPL10 R98S selective sensitivity to the clinically available Bcl-2 inhibitor Venetoclax (ABT-199) was supported by suppression of splenomegaly and the absence of human leukemia cells in the blood of T-ALL xenografted mice. These results shed new light on the oncogenic function of ribosomal mutations in cancer, provide a novel mechanism for BCL-2 upregulation in leukemia, and highlight BCL-2 inhibition as a novel therapeutic opportunity in RPL10 R98S defective T-ALL. ispartof: LEUKEMIA vol:33 issue:2 pages:319-332 ispartof: location:England status: published

Postnatal hematopoietic stem (and progenitor) cells (HS(P)Cs) are especially vulnerable to oxidative stress, leading to early hematopoietic senescence and/or malignant transformation. Elevated intracellular reactive oxygen species (ROS) can, among others, oxidize nucleotides, and thus can result in genotoxicity and mutagenesis if left unrepaired. Oxidized bases, as well as other spontaneous single base modifications, are recognized and repaired by the base excision repair (BER) pathway. Hence, the BER pathway is crucial to maintain genome integrity. In contrast to other DNA repair pathways however, the role of BER in maintaining HSPC functionality remains enigmatic, chiefly because knockout (KO) of BER genes is in many cases embryonic lethal. BER is a complex multi-step repair process. After initial removal and excision of the damaged base, the apurinic/apyrimidinic (AP) site is processed by the AP endonuclease (APEX1) enzyme. At this point, the BER pathway branches into 2 sub-pathways, namely the short-patch (SP-BER; wherein DNA polymerase beta (Polβ), Ligase III (Lig3) together with X-ray repair cross-complementing protein 1 (Xrcc1) are active) and the long-patch BER (LP-BER; wherein Lig1, Flap Structure-Specific Endonuclease 1 (Fen1), and sometimes Polβ are active) for the repair synthesis and the gap filling steps. In this study we wished to address the role of BER in adult hematopoiesis. Therefore, we used CRISPR-Cas9 to KO different BER genes in adult bone marrow (BM) HS(P)Cs, including two genes common to the BER (sub-)pathway(s) (Apex1 and Polβ) as well as one gene in the SP-BER (Xrcc1) and one gene in the LP-BER (Lig1) pathway. The effect thereof was evaluated on HS(P)C repopulation in vivo as well as on HS(P)C expansion during long-term in vitro culture (using the culture medium described by Wilkinson et al., Nature 2019). All CRISPR-Cas9 experiments were validated using a second sgRNA targeting the selected BER genes. Lig1-KO caused in vivo HSPC dysfunction: at 20 weeks post-transplantation, significantly less Lig1 KO cells were observed in the committed progenitor (HPC) and lineage committed (Lin +) BM compartments. By contrast, KO of Xrcc1 had only minor effects on HS(P)C repopulation, but we observed increased HSC expansion and myeloid biased differentiation in some recipient mice, which might correspond to clonal hematopoiesis and is consistent with the finding of XRCC1 loss-of-function mutation in myelodysplastic patients (Joshi et al, Ann Hematol 2016). Knockout of Polβ did not affect hematopoiesis in vivo or in vitro. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HS(P)Cs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less Apex1 deficient cells were detected in the different blood lineages and nearly no CRISPR-Cas9 KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of Apex1 KO BM cells was observed. APEX1 has two major functional activities, namely its nuclease activity, involved in BER, and its redox activity (also called Ref-1 function) important in reducing oxidized transcription factors and therefore implicated in transcriptional regulation. However, little is known regarding the nuclease and Ref-1 function(s) in primary adult hematopoietic cells. We therefore cultured BM HS(P)Cs for 1 week in the continuous presence of 2 distinct chemicals blocking the APEX1 nucleases, or 2 different chemicals inhibiting specifically the Ref-1 function. We demonstrated that both APEX1 functions are essential for hematopoiesis, even if the 2 functions appear to support the survival, expansion and maintenance of HS(P)Cs through different mechanisms. While the Ref-1 function was essential for proliferation (as both Ref-1 inhibitors cause cell cycle arrest) of all the lineages (including the Lin + cells), both inhibitors of the nuclease function affected more the expansion/survival of the less committed HS(P)Cs without leading to any cell cycle arrest. In conclusion, this study demonstrates for the first time the important role of BER genes in adult hematopoiesis, often deregulated in cancer, including hematopoietic malignancies. We observed a particularly severe phenotype upon loss of Apex1 in adult HSPCs, and ongoing studies (such as RNA sequencing analysis) should provide novel insights in underlying mechanisms of APEX1 deficiencies in HS(P)Cs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.