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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.

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 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.

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.

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.

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.

// Laura Fancello 1,* , Kim R. Kampen 1,* , Isabel J.F. Hofman 1 , Jelle Verbeeck 1 and Kim De Keersmaecker 1 1 KU Leuven-University of Leuven, Department of Oncology, LKI-Leuven Cancer Institute, Leuven, Belgium * These authors have contributed equally to this work Correspondence to: Kim De Keersmaecker, email: // Keywords : ribosomal protein, haploinsufficient tumor suppressor, TCGA, breast cancer Received : January 05, 2017 Accepted : January 11, 2017 Published : January 29, 2017 Abstract For many years, defects in the ribosome have been associated to cancer. Recently, somatic mutations and deletions affecting ribosomal protein genes were identified in a few leukemias and solid tumor types. However, systematic analysis of all 81 known ribosomal protein genes across cancer types is lacking. We screened mutation and copy number data of respectively 4926 and 7322 samples from 16 cancer types and identified six altered genes ( RPL5 , RPL11 , RPL23A , RPS5 , RPS20 and RPSA ). RPL5 was located at a significant peak of heterozygous deletion or mutated in 11% of glioblastoma, 28% of melanoma and 34% of breast cancer samples. Moreover, patients with low RPL5 expression displayed worse overall survival in glioblastoma and in one breast cancer cohort. RPL5 knockdown in breast cancer cell lines enhanced G2/M cell cycle progression and accelerated tumor progression in a xenograft mouse model. Interestingly, our data suggest that the tumor suppressor role of RPL5 is not only mediated by its known function as TP53 or c-MYC regulator. In conclusion, RPL5 heterozygous inactivation occurs at high incidence (11-34%) in multiple tumor types, currently representing the most common somatic ribosomal protein defect in cancer, and we demonstrate a tumor suppressor role for RPL5 in breast cancer.

Summary The anti-leukemia 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 triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression., Graphical Abstract, Highlights • SAMHD1-deficient cells die upon exposure to deoxyguanosine (dG) • dG induces apoptosis in cells, including cancer cells, lacking SAMHD1 • PNP-inhibitors such as forodesine and dG synergistically trigger cell death • dG and forodesine kill mutated leukemic cells without SAMHD1 expression, SAMHD1 degrades deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Davenne et al. find 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.

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.

Metabolic reprogramming is a well-established hallmark of cancer cells, which supports tumor growth, survival, and chemotherapy resistance. In this respect, a subset of cancers gets addicted to intracellular serine/glycine synthesis via overexpression of the enzymes PHGDH, PSAT1, PSPH or SHMT1/2. This cancer-specific dependency highlights a novel therapeutic opportunity, as normal cells solely rely on serine and glycine uptake from their environment. Well established examples of serine/glycine-synthesis addicted tumors are triple-negative breast cancer and T-cell leukemia (1, 2), which are both currently treated with toxic intensive chemotherapy regimens. In the past decade, researchers have attempted to repurpose approved drugs for cancer treatment, with the aim to develop less toxic low cost therapies that can rapidly enter the clinical practice. In this work, we made use of Candida albicans biofilms as a lower eukaryotic screening system that specifically upregulates expression of serine/glycine synthesis enzymes in response to sub-lethal stress. Using this platform, we discovered two repurposed compounds, sertraline and thimerosal, that show selective toxicity to serine/glycine synthesis dependent cancer cell lines, while they had no effect on cancer and normal lymphoid cell lines that take up serine and glycine from their environment. By using a combination of a PHGDH enzymatic activity assay, molecular docking and labeled serine tracing experiments, we discovered that sertraline and thimerosal inhibit serine/glycine synthesis enzymes SHMT and PHGDH respectively. In contrast to thimerosal, for which clinical applications are limited because of a toxic mercury group in its structure, sertraline is a widely used anti-depressant for which synergy with established anticancer drugs in breast cancer had already been described. Most notably, combining sertraline with a mitochondrial inhibitor, such as the clinically used antimalarial drug artemether, resulted in an even more pronounced inhibition of serine/glycine synthesis dependent proliferation, both in cultured cells and in an in vivo breast cancer xenograft model. Collectively, our study provides molecular insights into the repurposed mode-of-action of sertraline and allows to delineate a hitherto unidentified group of cancers that are particularly sensitive to treatment with sertraline, including breast cancers and leukemias. References: 1. R. Possemato, et al., Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature 476, 346–350 (2011). 2. K. R. Kampen, et al., Translatome analysis reveals altered serine and glycine metabolism in T-cell acute lymphoblastic leukemia cells. Nature Communications 10, 2542 (2019). Citation Format: Shauni L. Geeraerts, Kim R. Kampen, Gianmarco Rinaldi, Purvi Gupta, Mélanie Planque, Kaat De Cremer, Katrijn De Brucker, Stijn Vereecke, Benno Verbelen, Pieter Vermeersch, David Cassiman, Sarah-Maria Fendt, Arnout Voet, Bruno P.A. Cammue, Karin Thevissen, Kim De Keersmaecker. Repurposing the anti-depressant sertraline to target serine/glycine synthesis addicted cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1789.

Supplemental Digital Content is available in the text. ispartof: Hemasphere vol:2 issue:2 ispartof: location:United States status: published

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. ispartof: CANCER RESEARCH vol:78 issue:20 pages:5940-5948 ispartof: location:United States status: published

Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10-20 protein-altering lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34+CD38- multipotent progenitor cells for four T-ALL cases. Hierarchical clustering detected a dominant leukemia cluster at diagnosis, accompanied by a few smaller clusters harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) among various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34+CD38- cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single-cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell. ispartof: LEUKEMIA vol:32 issue:6 pages:1358-1369 ispartof: location:England status: published

ispartof: HEMASPHERE vol:3 issue:6 ispartof: location:United States status: published

The NUP214-ABL1 fusion protein is a constitutively active protein tyrosine kinase that is found in 6% of patients with T-cell acute lymphoblastic leukemia and that promotes proliferation and survival of T-lymphoblasts. Although NUP214-ABL1 is sensitive to ABL1 kinase inhibitors, development of resistance to these compounds is a major clinical problem, underlining the need for additional drug targets in the sparsely studied NUP214-ABL1 signaling network. In this work, we identify and validate the SRC family kinase LCK as a protein whose activity is absolutely required for the proliferation and survival of T-cell acute lymphoblastic leukemia cells that depend on NUP214-ABL1 activity. These findings underscore the potential of SRC kinase inhibitors and of the dual ABL/SRC kinase inhibitors dasatinib and bosutinib for treating of NUP214-ABL1 positive T-cell acute lymphoblastic leukemia. In addition, we used mass spectrometry to identify protein interaction partners of NUP214-ABL1. Our results strongly support that the signaling network of NUP214-ABL1 is distinct from that previously reported for BCR-ABL1. Moreover, we identify three NUP214-ABL1 interacting proteins, MAD2L1, NUP155, and SMC4, as strictly required for the proliferation and survival of NUP214-ABL1 positive T-cell acute lymphoblastic leukemia cells. In conclusion, this work identifies LCK, MAD2L1, NUP155 and SMC4 as four new potential drug targets in NUP214-ABL1 positive T-cell acute lymphoblastic leukemia. ispartof: Haematologica vol:99 issue:1 pages:85-93 ispartof: location:Italy status: published

Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor associated with poor outcome. In this study, we analyzed the prognostic relevance of genetic alterations, immunophenotypic markers, and microarray gene expression signatures in a panel of 53 adult T-ALL patients treated in the Eastern Cooperative Oncology Group E2993 clinical trial. An early immature gene expression signature, the absence of bi-allelic TCRG deletion, CD13 surface expression, heterozygous deletions of the short arm of chromosome 17, and mutations in IDH1/IDH2 and DNMT3A genes are associated with poor prognosis in this series. In contrast, expression of CD8 or CD62L, homozygous deletion of CDKN2A/CDKN2B, NOTCH1 and/or FBXW7 mutations, and mutations or deletions in the BCL11B tumor suppressor gene were associated with improved overall survival. Importantly, the prognostic relevance of CD13 expression and homozygous CDKN2A/CDKN2B deletions was restricted to cortical and mature T-ALLs. Conversely, mutations in IDH1/IDH2 and DNMT3A were specifically associated with poor outcome in early immature adult T-ALLs. This trial was registered at www.clinicaltrials.gov as #NCT00002514.

SummaryConstitutive phosphoinositide 3-kinase (PI3K)/Akt activation is common in T cell acute lymphoblastic leukemia (T-ALL). Although four distinct class I PI3K isoforms (α, β, γ, δ) could participate in T-ALL pathogenesis, none has been implicated in this process. We report that in the absence of PTEN phosphatase tumor suppressor function, PI3Kγ or PI3Kδ alone can support leukemogenesis, whereas inactivation of both isoforms suppressed tumor formation. The reliance of PTEN null T-ALL on the combined activities of PI3Kγ/δ was further demonstrated by the ability of a dual inhibitor to reduce disease burden and prolong survival in mice as well as prevent proliferation and promote activation of proapoptotic pathways in human tumors. These results support combined inhibition of PI3Kγ/δ as therapy for T-ALL.

The TLX1 transcription factor oncogene is frequently activated by chromosomal translocations in T-cell acute lymphoblastic leukemia (T-ALL) and defines a distinct molecular group of tumors characterized by differentiation arrest at the early cortical stage of thymocyte differentiation and excellent response to therapy. Recent developments from the analysis of genomic data on TLX1-specific transcriptional targets and analysis of the molecular mechanisms of TLX1 transformation in human- and mouse-induced leukemias have shown novel insight into the activity of this transcription factor oncogene. Aberrant expression of TLX1 in T-cell progenitors disrupts normal T-cell development and triggers the development of aneuploidy during T-cell transformation. Importantly, the disruption of the mitotic checkpoint in TLX1-induced tumors may be linked not only to the acquisition of secondary genetic alterations in T-ALL but also to increased sensitivity of these tumors to chemotherapy with drugs targeting the formation of the mitotic spindle. Clin Cancer Res; 17(20); 6381–6. ©2011 AACR.

Background Activating NOTCH1 mutations are common in T-cell acute lymphoblastic leukemia. Inhibition of NOTCH1 signaling with γ-secretase inhibitors causes cell cycle block, but only after treatment for several days. We further documented the effects of γ-secretase inhibitor treatment on T-cell acute lymphoblastic leukemia cell lines and tested whether combining γ-secretase inhibitors with other anti-cancer drugs offers a therapeutic advantage. Design and Methods The effect of γ-secretase inhibitor treatment and combinations of γ-secretase inhibitors with chemotherapy or glucocorticoids was assessed on T-cell acute lymphoblastic leukemia cell lines. We sequenced NOTCH1 in T-cell acute lymphoblastic leukemia cases with ABL1 fusions and tested combinations of γ-secretase inhibitors and the ABL1 inhibitor imatinib in a T-cell acute lymphoblastic leukemia cell line. Results γ-secretase inhibitor treatment for 5–7 days reversibly inhibited cell proliferation, caused cell cycle block in sensitive T-cell acute lymphoblastic leukemia cell lines, and caused differentiation of some T-cell acute lymphoblastic leukemia cell lines. Treatment for 14 days or longer was required to induce significant apoptosis. The cytotoxic effects of the chemotherapeutic agent vincristine were not significantly enhanced by addition of γ-secretase inhibitors to T-cell acute lymphoblastic leukemia cell lines, but γ-secretase inhibitor treatment sensitized cells to the effect of dexamethasone. NOTCH1 mutations were identified in all T-cell acute lymphoblastic leukemia patients with ABL1 fusions and in a T-cell acute lymphoblastic leukemia cell line expressing NUP214-ABL1 . In this cell line, the anti-proliferative effect of imatinib was increased by pre-treatment with γ-secretase inhibitors. Conclusions Short-term treatment of T-cell acute lymphoblastic leukemia cell lines with γ-secretase inhibitors had limited effects on cell proliferation and survival. Combinations of γ-secretase inhibitors with other drugs may be required to obtain efficient therapeutic effects in T-cell acute lymphoblastic leukemia, and not all combinations may be useful.

International audience; Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

The BCR-ABL1 fusion kinase is frequently associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia but is rare in T-cell acute lymphoblastic leukemia (T-ALL). We recently identified NUP214-ABL1 as a variant ABL1 fusion gene in 6% of T-ALL patients. Here we describe the identification of another ABL1 fusion, EML1-ABL1, in a T-ALL patient with a cryptic t(9;14)(q34;q32) associated with deletion of CDKN2A (p16) and expression of TLX1 (HOX11). Echinoderm microtubule-associated protein-like 1-Abelson 1 (EML1-ABL1) is a constitutively phosphorylated tyrosine kinase that transforms Ba/F3 cells to growth factor-independent growth through activation of survival and proliferation pathways, including extracellular signal-related kinase 1/2 (Erk1/2), signal transducers and activators of transcription 5 (Stat5), and Lyn kinase. Deletion of the coiled-coil domain of EML1 abrogated the transforming properties of the fusion kinase. EML1-ABL1 and breakpoint cluster region (BCR)-ABL1 were equally sensitive to the tyrosine kinase inhibitor imatinib. These data further demonstrate the involvement of ABL1 fusions in the pathogenesis of T-ALL and identify EML1-ABL1 as a novel therapeutic target of imatinib. (Blood. 2005;105:4849-4852)

Over 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL) harbor activating mutations in Notch1 gene, making this receptor a promising therapeutic target for T-ALL. Υ-secretase inhibitors (GSIs), which block a proteolytic cleavage required for NOTCH1 activation, have been clinically explored for the treatment of T-ALL. However, lack of primary responses and relapses have prevented further development of GSIs in the clinic, highlighting the need to identify novel therapies synergistic with NOTCH1 inhibition for the treatment of highly aggressive NOTCH1-mutant T-ALL. To identify new antileukemic drugs for T-ALL we designed an expression-based drug screen utilizing drug profiles contained in the Connectivity Map (cMAP) to search for positive connections with gene signatures driven by NOTCH1 inhibition in this disease. This analyses uncovered 17 compound candidates for synergistic activity with NOTCH1 inhibition, including drugs with known mechanism of action: inhibitors of the mTOR/PI3K/AKT pathway and histone deacetylase inhibitors; as well as compounds with unrelated mechanisms, pyrvinium pamoate (antihelmintic) and withaferin A (antiangiogenic). Pair-wise gene set enrichment analyses on the expression profile driven by each of these drugs in T-ALL cells revealed compounds with transcriptional programs partially overlapping those driven by PI3K/AKT inhibitors, and drugs with distinctive expression profiles, such as withaferin A, suggestive of a unique mode of action. We next characterized the activity of each of the cMAP hits alone and in combination with the GSI DBZ in vitro using a panel of 5 NOTCH1-mutant T-ALL cell lines (CUTLL1, DND41, HPBALL, RPMI and CCRF-CEM). In this analysis, 5 drugs scored as highly active (IC50 Functional annotation and DeMAND algorithm analyses on the expression profile of withaferin A revealed downregulation of genes and pathways involved in protein translation and identified 21 components of the ribosome and the translation machinery as potential effectors of the antitumoral activity of the drug in T-ALL, including two subunits of the eIF2A translation complex (eIF2S1 and eIF2S2) which controls translation initiation via phosphorylation of the eIF2S1 subunit under conditions of cellular stress. Polysome profiling, nascent protein, and Western blot analyses of cells treated with withaferin A confirmed an inhibitory effect of the drug in protein translation mediated by eIF2S1 phosphorylation. Consistently, expression of a non-phosphorylable mutant form of eIF2S1 in leukemia cell lines rescued the effects of withaferin A in vitro. Our results illustrate the power of our expression-based and in vivo validation approach toward the identification of antitumor drug combinations for the treatment of human cancer. Citation Format: Marta Sanchez-Martin, Alberto Ambesi-Impiombato, Yue Qin, Daniel Herranz, Mukesh Bansal, Tiziana Girardi, Elisabeth Paietta, Martin S. Tallman, Jacob M. Rowe, Kim De Keersmaecker, Andrea Califano, Adolfo Ferrando. Synergistic antileukemic therapies in NOTCH1-induced T-ALL [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 49.

The association between ribosome defects and cancer became clear with the recent discovery of somatic mutations in ribosomal protein genes in several cancers, such as lymphoid leukemia’s and glioblastoma (Nat Genet. 2013 Feb;45(2):186-90 & Blood. 2016 Feb 25;127(8):1007-16 & Nature. 2014 Jan 23;505(7484):495-501). To further delineate the role of ribosomal proteins in human cancer, we systematically screened the TCGA database for genetic lesions in ribosomal protein genes, confirmed the contributing capacity of the most common somatic ribosomal protein defect in cancer pathogenesis by molecular interference, and explored the mode of action of this ribosomal protein defect. Systematic analysis of TCGA mutation and copy number data of respectively 4926 and 7322 patients representing 16 cancer types for defects in all 81 ribosomal protein genes. After stringent filtering, six ribosomal protein encoding genes (RPL5, RPL11, RPL23A, RPS5, RPS20 and RPSA) were significantly altered and identified as candidate cancer driver genes. RPL5 was located at a significant peak of heterozygous deletion on chromosome 1p22 and showed significant mutations and deletions in 11% of glioblastoma (GBM), 28% of melanoma, 34% of breast cancer (BRCA), and in 20% of multiple myeloma cases, thereby supporting heterozygous RPL5 inactivation to be the most common somatic ribosomal protein defect in human cancer. Human specific RPL5 doxycycline inducible knockdown (sh-hRPL5) in TP53 WT and TP53 mutant human breast cancer cell lines (MCF7 and MDA-MB-231) and in a TP53 mutant human glioblastoma cell line (U-118 MG) proved that RPL5 knockdown accelerated in vivo tumor progression in NSG mice. This acceleration was associated with reduced phosphorylation of CDK1tyr15, which is required for cell cycle progression from G2 to mitosis. Whereas RPL5 has been implicated in TP53 and MYC regulation, no consistent effects of RPL5 downregulation on these proteins could be detected in TP53 WT and TP53 homozygous R280K mutant breast cancer models (sh-hRPL5). Interestingly, partial RPL5 inactivation was found to be associated with PTEN protein suppression in these tumors. This observation was confirmed in various cell models; in normal mouse neural stem/progenitor cells and mouse bone marrow cells (RPL5+/lox by INFβ and poly I:C), and also in GBM cancer pre-disposition mouse neural stem/progenitor cell model (sh-mRPL5 TP53 -/-). RPL5 downregulation also accelerated tumor formation in the PTEN deficient GBM cell line U-118 MG, underscoring undefined PTEN-independent mechanisms of RPL5. Overall, we identified RPL5 as a new tumor suppressor that shows heterozygous inactivation in 11-34% of multiple human cancer types. Partial molecular inactivation of RPL5 supported a tumor suppressor function for RPL5 in accelerating breast cancer and glioblastoma progression in vivo, which may be, in part, due to a new function of RPL5 in regulating PTEN. Citation Format: Kim R. Kampen, Laura Fancello, Jelle Verbeeck, Kim De Keersmaecker. Contribution of heterozygous loss of ribosomal protein L5 as general tumor suppressor in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1546. doi:10.1158/1538-7445.AM2017-1546

The JAK-STAT pathway is one of the major mechanisms through which cellular proliferation, differentiation, migration, survival and apoptosis are regulated in response to external stimuli. Promiscuous activation of this pathway is a major driver in the pathogenesis of myeloproliferative neoplasms, often characterized by clonal expansion of mature myeloid cells. The JAK2-V617F allele is the most common and well characterized mutation linked to this class of leukemias. A change of G to T at nucleotide 1849 of the coding sequence (CDS) results in substitution of valine to phenylalanine at codon 617. JAK2-V617F mutant cells have strongly increased activation of JAK-STAT signaling, which can partially be explained by its ability abolish inhibition of kinase activity and increase the Km of the enzyme. It is however unclear if these effects are sufficient to explain the strong activation of the JAK-STAT pathway induced by the V617F mutation. Here, we demonstrate that this mutation destabilizes a programmed -1 ribosomal frameshift (-1 PRF) signal in the JAK2 mRNA, leading to decreased rates of -1 PRF promoted by this element. There is an inverse relationship between -1 PRF and mRNA stability. Thus, decreased -1 PRF results in increased abundance of the JAK2 mRNA and increased JAK2 protein expression. Prior studies established that elevated expression levels of JAK proteins are sufficient to transform cells in vitro, suggesting that -1 PRF may normally help to limit JAK2 expression. Silent protein coding changes in the -1 PRF signal slippery sequence ablating JAK2 frameshifting also increased JAK2 expression and phenocopied the transforming activity of JAK2-V617F in cell culture. Further, the combination of the V617F and slippery site mutations conferred an additive effect on cellular transformation. The presence at least three additional -1 PRF signals in the JAK2 mRNA suggests that -1 PRF plays a significant role in controlling JAK2 expression. Citation Format: Yousuf A. Khan, Sergey O. Sulima, Joe Kendra, Vivek M. Advani, John E. Jones, Joseph Briggs, Kim de Keersmaecker, Jonathan D. Dinman. A programmed ribosomal frameshifting defect potentiates the transforming activity of the JAK2-V617F mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3034. doi:10.1158/1538-7445.AM2017-3034

The mutational landscape of T-cell acute lymphoblastic leukemias (T-ALL) has been intensively studied over the past decades and we now have an extensive list of genetic defects that can occur in these tumors.[1][1] Central to T-ALL pathogenesis is the NOTCH1 signaling cascade which is hyperactivated

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling1. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2) 2,3, in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) 4 by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation. © 2012 Nature America, Inc. All rights reserved., J.N. was supported by the Damon Runyon Cancer Research Foundation. I.A. was supported by the US National Institutes of Health (RO1CA133379, RO1CA105129, R21CA141399, RO1CA149655 and RO1GM088847), The Leukemia & Lymphoma Society, The V Foundation, the American Cancer Society (RSG0806801) and the Dana Foundation. The Aifantis laboratory is also supported by a Feinberg Lymphoma Pilot grant. This study was also supported by the Fund for Scientific Research of Flanders (P.V.V. and K.D.K.); the US National Library of Medicine (1R01LM010140-01 to R.R.); the Eastern Cooperative Oncology Group tumor bank; a Northeast Biodefense Center American Recovery and Reinvestment Act award (U54-AI057158 to R.R.); the US National Institutes of Health (R01CA120196 and R01CA155743 to A.F.); the Stand Up To Cancer Innovative Research Award (A.F.), the Chemotherapy Foundation (I.A. and A.F.); the Rally Across America Foundation (A.F.) and the Swim Across America Foundation (A.F.). P.V.V. is an American Society of Hematology Scholar, and I.A. and A.F. are Leukemia & Lymphoma Society Scholars. M.D. is supported by grants from Spanish Ministerio de Ciencia e Innovación (BFU2009-09074 and MECCONSOLIDER CSD2007-00023), Generalitat Valenciana (PROMETEO2006/134) and an EU Research Grant (UE-HEALH-F2-2008-201666). F.P. is supported by the Institut du Cancer, the Association Laurette Fugain, the Ligue National Contre le Cancer and also by INSERM, CEA and StemPole. S.P. is supported by a fellowship by the Institut du Cancer. I.A. is a Howard Hughes Medical Institute Early Career Scientist.

Inhibition of the BCR-ABL1 tyro-sine kinase with selective small molecule tyrosine kinase inhibitors such as imatinib, nilotinib or dasatinib has revolutionized the treatment of chronic myeloid leukemia (CML).[1][1],[2][2],[3][3] These drugs target the ATP-binding pocket of the ABL1 kinase domain

The protein tyrosine phosphatase CD45, encoded by the PTPRC gene, is well known as a regulator of B- and T-cell receptor signaling. In addition, CD45 negatively regulates JAK family kinases downstream of cytokine receptors. Here, we report the presence of CD45 inactivating mutations in T-cell acute lymphoblastic leukemia. Loss-of-function mutations of CD45 were detected in combination with activating mutations in IL-7R, JAK1, or LCK, and down-regulation of CD45 expression caused increased signaling downstream of these oncoproteins. Furthermore, we demonstrate that down-regulation of CD45 expression sensitizes T cells to cytokine stimulation, as observed by increased JAK/STAT signaling, whereas overexpression of CD45 decreases cytokine-induced signaling. Taken together, our data identify a tumor suppressor role for CD45 in T-cell acute lymphoblastic leukemia.

To the editor: Recent data indicate that NOTCH1 mutations significantly increase the risk of CLL progression toward Richter syndrome (RS) and chemoresistance,[1][1],[2][2] and that activation of NOTCH1 at time of CLL diagnosis is an independent prognostic factor of poor survival.[1][1],[3][3] We

The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL)(1,2). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

SummaryImatinib has revolutionized the treatment of Bcr-Abl1+ chronic myeloid leukemia (CML), but, in most patients, some leukemia cells persist despite continued therapy, while others become resistant. Here, we report that PlGF levels are elevated in CML and that PlGF produced by bone marrow stromal cells (BMSCs) aggravates disease severity. CML cells foster a soil for their own growth by inducing BMSCs to upregulate PlGF, which not only stimulates BM angiogenesis, but also promotes CML proliferation and metabolism, in part independently of Bcr-Abl1 signaling. Anti-PlGF treatment prolongs survival of imatinib-sensitive and -resistant CML mice and adds to the anti-CML activity of imatinib. These results may warrant further investigation of the therapeutic potential of PlGF inhibition for (imatinib-resistant) CML.

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17-92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17-92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17-92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of T-cell precursors that mainly occurs in children and adolescents. A variety of oncogenic events that are involved in the pathogenesis of T-ALL have been identified, including NOTCH1 and PTEN mutations, overexpression of TAL1 , LYL1 and

Genetic alterations causing constitutive tyrosine kinase activation are observed in a broad spectrum of cancers. Thus far, these mutant kinases have been localized to the plasma membrane or cytoplasm, where they engage proliferation and survival pathways. We report that the NUP214-ABL1 fusion is unique among these because of its requisite localization to the nuclear pore complex for its transforming potential. We show that NUP214-ABL1 displays attenuated transforming capacity as compared to BCR-ABL1 and that NUP214-ABL1 preferentially transforms T cells, which is in agreement with its unique occurrence in T cell acute lymphoblastic leukemia. Furthermore, NUP214-ABL1 differs from BCR-ABL1 in subcellular localization, initiation of kinase activity, and signaling and lacks phosphorylation on its activation loop. In addition to delineating an unusual mechanism for kinase activation, this study provides new insights into the spectrum of chromosomal translocations involving nucleoporins by indicating that the nuclear pore context itself may play a central role in transformation. ispartof: Molecular cell vol:31 issue:1 pages:134-142 ispartof: location:United States status: published

Abstract 471 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of oncogenic transcription factors. Thus, 5% to 10% of pediatric and up to 30% of adult T-ALL patients show aberrant expression of the TLX1 transcription factor oncogene. Aberrant expression of TLX1 in Lck-TLX1 transgenics induces transformation of T-cell progenitors and the development of clonal T-cell lymphoblastic tumors with over 80% penetrance and a latency of 29 weeks. The prolonged latency before T-ALL development in TLX1 transgenic mice and the clonal nature of these tumors suggested the presence of cooperating mutations involved in the pathogenesis of these leukemias. Array Comparative Genomic Hybridization (aCGH) analysis identified several recurring chromosomal alterations in these tumors including three heterozygous deletions on chromosome 12, with a common deleted region containing only the Bcl11b gene. Moreover, DNA sequence analysis of Bcl11b showed the presence of Bcl11b mutations in 4/15 (27%) mouse TLX1 induced T-ALLs, which together with the 3 Bcl11b deletions identified in our aCGH analysis brings the prevalence of Bcl11b alterations in mouse TLX1 induced tumors to 7/15 (47%). Notably, mutation analysis of Bcl11b in a panel of 21 non TLX1-transgenic mouse T-ALL tumors failed to detect any Bcl11b mutations (P Bcl11b encodes a zinc finger transcription factor with a critical role in the differentiation and survival of T-cell progenitors in the thymus. Given the similarities between our mouse model of TLX1-induced leukemia and human T-ALL, we hypothesized that BCL11B alterations could also be implicated in the pathogenesis of human T-ALL. Most notably, aCGH analysis of human T-ALL samples showed the presence of focal heterozygous deletions encompassing the BCL11B locus in 2/69 (3%) T-ALL cases. In addition, mutation analysis of BCL11B in human T-ALL demonstrated the presence of somatic truncating frameshift mutations and somatic missense point mutations in 9/71 (13%) cases analyzed. Interestingly, all 6 BCL11B missense mutations involved zinc finger domains. One sample showed compound heterozygous BCL11B mutations while the remaining cases harbored heterozygous BCL11B lesions. Finally, expression analysis of TLX1 or TLX3 in 8 T-ALLs harboring mutations or deletions in BCL11B with RNA available showed aberrant expression of these transcription factor oncogenes in 5/8 (63%) [TLX1 (4/8); TLX3 (1/8)] of these samples. The identification of a high prevalence of Bcl11b mutations and deletions in TLX1-induced tumors suggested a specific genetic interaction between these two genes in T-cell transformation. We contemplated two alternative scenarios. First, TLX1 could induce partial inactivation of BCL11B in T-cell transformation, an effect that would be reinforced by secondary genetic alterations in the BCL11B locus during tumor progression. Alternatively, BCL11B mutations and TLX1 overexpression could operate in parallel pathways that contribute to distinct but complementary aspects of tumor development. To test if BCL11B could be a direct transcriptional target of TLX1 in T-ALL, we analyzed the binding of TLX1 to the BCL11B promoter via chromatin immunoprecipitation analysis in ALL-SIL, a T-ALL cell line expressing high levels of TLX1 as result of the t(10;14)(q24;q11.2) translocation. This analysis revealed that indeed TLX1 binds to the BCL11B promoter. Moreover, siRNA knockdown of TLX1 in this cell line resulted in transcriptional upregulation of BCL11B, indicating that BCL11B is a direct transcriptional target downregulated by TLX1 in T-ALL. The model that emerges from these results is that the TLX1 oncogene directly downregulates the BCL11B tumor suppressor gene, and that the oncogenic effects of this transcriptional regulatory axis can be fixed and reinforced by secondary deletions and mutations in the BCL11B locus acquired during tumor progression. Altogether, these results highlight the power of integrated genomic analysis of human and mouse leukemias, identify a TLX1-BCL11B transcriptional regulatory axis in the pathogenesis of T-ALL and define BCL11B as a tumor suppressor gene recurrently deleted and mutated in 16% of human T-ALL patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 141 Introduction: T-cell lymphoblastic leukemia (T-ALL) arises from clonal expansion of a lymphoid progenitor that has undergone stepwise alteration at distinct stages of differentiation. It is suggested that a set of cooperative mutations that affect different pathways are required before thymocytes become fully malignant. Despite major improvements in our understanding of the molecular genetics of T-ALL, the underlying mechanisms that lead to the abnormal proliferation and enhanced survival of the leukemic cells remain largely unknown. Results: Array CGH analysis revealed an acquired homozygous microdeletion at chromosome 18p11 in 6 % of T-ALL cases. The deleted region was only 125 kb in size and restricted to the PTPN2 (protein tyrosine phosphatase, non-receptor type 2) locus. PTPN2 encodes an intracellular non-transmembrane tyrosine-specific phosphatase that functions as a negative regulator of a variety of signaling proteins including several members of the janus kinase (JAK) and of signal transducer and activator of transcription (STAT) families, growth factor receptors and SRC family kinases. Homozygous deletion of PTPN2 was specifically found in cases with aberrant expression of the TLX1 transcription factor, with two cases also harboring the NUP214-ABL1 fusion. Analysis of additional TLX1 positive cases by quantitative PCR identified loss of one copy of PTPN2 in 5 out of 20 cases. No mutations were detected in the coding region of PTPN2. To determine the effect of loss of PTPN2 in T-cells, we downregulated the expression of PTPN2 using RNAi technology. siRNA mediated knock-down of PTPN2 affected activation of JAK1 associated cytokine receptors implicated in T-cell development. Ligand stimulation of IL7 and interferon gamma receptor resulted in an augmented and prolonged phosphorylation of JAK1 as well as downstream targets STAT1 and STAT5 in T-ALL cell lines with knock-down of PTPN2. In addition, knock- down of Ptpn2 sensitized the pro B-cell line Ba/F3 to transformation by wild type JAK1 confirming a clear relationship between loss of PTPN2 and JAK1 activation. Knock-down of PTPN2 expression also provided a proliferative advantage and reduced sensitivity to kinase inhibitors in lymphoblastic leukemia cell lines HSB-2 and ALL-SIL. Conclusion: In conclusion, our data provide genetic and functional evidence for a tumor suppressor role of PTPN2 in T-ALL and warrant testing of JAK inhibitors for the treatment of this specific subset of T-ALLs as well as further analysis of a potential negative impact of loss of PTPN2 on responsiveness to anti-cancer treatments. Disclosures: Ferrando: Merck, Pfizer: Research Funding.

Abstract 42 Although BCR-ABL kinase inhibitors (TKIs) have revolutionized treatment of CML, they do not eradicate disease and treatment resistance can emerge. We recently identified Placental Growth Factor (PlGF) as an additional pathogenic factor in murine BCR-ABL1+ leukemia. PlGF induces proliferation of bone marrow stromal cells and of BCR-ABL1+ cell lines. PlGF is upregulated in blood (PB) and bone marrow (BM) of mice with BCR-ABL1+ leukemia and it's inhibition by a monoclonal antibody (αPlGF) significantly prolongs survival of mice bearing BCR-ABL1-unmutated and -T315I mutant blast crisis (BC) CML (Loges et al., Blood 2008; 112: abstract 1094). Now, we have further characterized the role of PlGF in disease progression, the mechanism of action of αPlGF and validated PlGF as novel target in human CML. We first quantified PB and BM PlGF protein levels at different time points after inoculation of BCR-ABL1 transduced BM cells and found significantly increasing PlGF levels upon disease progression. In line with those data, PB and BM PlGF protein levels correlated with numbers of GFP-marked leukemia cells (r=0.81; p=7-fold more PlGF than leukemia cells (p=0.003), which corroborates our preclinical data and extends the concept that PlGF is primarily produced by stromal cells in CML patients. In summary inhibition of PlGF may serve as a new candidate to be targeted in combination therapies or in TKI refractory CML. Disclosures: Carmeliet: Thrombogenics: Research Funding.

Abstract 142 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of transcription factor oncogenes. TLX1/HOX11 was originally isolated from the recurrent t(10;14)(q24;q11) in T-ALL and is aberrantly expressed in 5% to 10% of pediatric and up to 30% of adult T-ALL cases. Tlx1 plays an important role during embryonic development and acts as a master transcriptional regulator necessary for the genesis of the spleen. TLX1 positive T-ALLs have a distinct gene expression profile resembling that of thymocytes blocked at the early double positive stage of development. This observation supports the hypothesis that aberrant expression of TLX1 contributes to the pathogenesis of T-ALL by interfering with critical transcriptional regulatory networks involved in cell proliferation, differentiation and survival during T-cell development. However, the identity of such oncogenic pathways and the mechanisms though which they operate are still largely unknown. In order to gain further insight into the mechanisms of transformation induced by TLX1, we generated a transgenic model of TLX1 induced T-ALL. In this model, a human TLX1 cDNA was expressed in developing thymocytes under the control of the proximal LCK promoter. TLX1 transgenic mice displayed a specific defect in T-cell development characterized by reduced thymic size and cellularity with increased apoptosis and a differentiation arrest at the CD4/CD8 double negative, CD25/CD44 double positive stage of differentiation (DN2 thymocytes). Long term follow up revealed that TLX1 transgenic mice develop tumors with a median latency of 29 weeks. TLX1 induced leukemias are characterized by increased thymic size and diffuse infiltration of bone marrow, spleen and peripheral organs by lymphoblasts expressing T-cell markers. Analysis of TCRβ expression and transplantation into isogenic recipients demonstrated that TLX1 tumors are clonal and transplantable. Microarray gene expression profiling of mouse and human T-ALLs showed that tumors from TLX1 transgenic mice have a gene expression signature that is highly related to that of human T-ALLs with aberrant expression of TLX1. Moreover, ChIP-on-chip analysis of promoters bound by TLX1 showed this genetic program is dominated by the downregulation of TLX1 direct target genes. Mutation profiling of T-cell oncogenes and array CGH analysis in mouse TLX1 tumors demonstrated the presence of cooperative mutations including Pten deletions, activating mutations in Notch1 and loss of Bcl11b. Strikingly, SKY analysis and array CGH revealed that 80% of TLX1 induced tumors had numerical chromosomal abnormalities including a high frequency of trisomy 15. Analysis of gene expression of ChIP-on-chip TLX1 direct target genes in double negative thymocytes from preleukemic mice showed downregulation of genes primarily involved in the control of chromosomal segregation during mitosis such as Bub1, Brca2, Chek1, Kntc1, Kif23, CenpE and Plk1. These data suggest that aberrant TLX1 expression directly promotes aneuploidy and contributes to T-cell transformation by interfering with the expression of genes responsible for chromosomal segregation during mitosis. Importantly, T-cell lymphoblasts from TLX1 transgenic mice failed to undergo a mitotic cell cycle arrest after treatment with taxol, a cell cycle inhibitor that interferes with microtubule remodeling during mitosis. Strikingly, karyotype analysis of a series of 59 pediatric T-ALLs demonstrated a high frequency of chromosomal gains and losses in TLX1 and TLX3 positive human T-cell tumors compared with other genetic subgroups of T-ALL (P Disclosures: Ferrando: Merck, Pfizer: Research Funding.

Introduction of imatinib and second-generation BCR/ABL inhibitors has revolutionized treatment of patients with Philadelphia chromosome positive (Ph+) leukemia, but leukemia cells persist even in successfully treated patients, and some patients develop resistance and ultimately relapse. The reasons for these drawbacks are not entirely resolved, but besides BCR/ABL the host stroma potentially plays an important (independent) role. Placental Growth Factor (PlGF), a homologue of VEGF, was already proven to be abundantly secreted by stromal cells in solid tumors. Therefore, we investigated the role of PlGF, and the therapeutic potential of αPlGF, a monoclonal antibody against PlGF, which we recently reported to have a broad anti-tumoral potential in pre-clinical models of solid tumors (Fischer et al., Cell, 2007), in BCR/ABL+ lymphoid and myeloid leukemia. First, we studied expression of PlGF by 5 different human and murine BCR/ABL+ leukemia cell lines (Bv-173, BaF3, 32D, K562, KCL22) in vitro and found that neither of the cell lines secreted PlGF protein, but expressed its target receptor VEGFR-1. In contrast, primary murine adherent bone marrow stromal cells (BMDSC) expressed abundant amounts of PlGF protein (up to 105 pg/ml/105 cells), indicating a potential stroma-related function of PlGF. Second, we analyzed whether PlGF could induce proliferation and thereby revealed dose-dependent induction of proliferation by recombinant PlGF in all analyzed leukemia cell lines. This pro-proliferative effect of PlGF was nearly completely abrogated by both αPlGF and an extracellular anti-VEGFR-1 antibody, thus indicating that it is mediated primarily by VEGFR-1. Third, we studied potential paracrine interactions between BMDSCs and leukemia cells by performing co-culture experiments. Remarkably, coculture of BMDSC with leukemia cells significantly induced proliferation of both cell types. We hypothesized, that this induction of proliferation might be mediated by PlGF and indeed found its nearly complete abrogation upon addition of αPlGF to the co-cultures. Furthermore, BMDSCs significantly upregulated PlGF secretion (2.1 fold; N=3; P=0.005) when cultured in presence of leukemia cells. Thus, we conclude, that stromal derived PlGF promotes proliferation of leukemia cells in a paracrine fashion and at the same time acts as autocrine pro-proliferative signal for stromal cells. To test this hypothesis in vivo, we established 3 different murine models of BCR/ABL+ myeloid and lymphoid leukemia. Subsequently, we analyzed PlGF protein as present in blood and bone marrow of diseased mice in comparison to healthy mice, and detected no PlGF protein in the peripheral blood of healthy mice and low amounts of PlGF protein in their bone marrow. In contrast, leukemic mice showed PlGF protein (76.5 ± 18.4 pg / ml plasma; N=7) in their circulation at levels comparable to mice bearing solid tumors, and, interestingly more than 8.9 fold (N=7; P

Leukemias are often associated with aberrant tyrosine kinase activity that occurs as a result of chromosomal translocations. These mutations are able to confer a proliferative and survival advantage to leukemic cells, as well as cooperate with other mutations that impair cell differentiation, thus leading to the development of leukemia. NUP214-ABL1 is one such recently identified fusion gene that is generated by episomal amplification. The presence of the fusion was recently identified in approximately 6% of patients with T-cell acute lymphoblastic leukemia (T-ALL). By the use of a murine retroviral bone marrow transplantation model we have demonstrated that mice transplanted with NUP214-ABL1 transduced bone marrow cells developed either a myeloproliferative disorder (MPD) with a disease latency of 70 to 118 days or a T cell lymphoblastic leukemia with a disease latency of 115 to 124 days. The myeloproliferative phenotype was characterized by splenomagaly and leukocytosis, and analysis of the histopathology revealed extramedullary hematopoiesis in the liver, lung, kidney and Peyer’s patches, and an increase of peripheral blood neutrophils. Flow cytometry of single cell suspensions from spleen and bone marrow samples of mice with a myeloproliferative phenotype demonstrated an increase of Gr-1+/Mac-1+ cells (approximately 70%). Two of the mice that were transplanted with NUP214-ABL1 transduced bone marrow cells developed T cell lymphomas that were characterized by large thymomas, a phenotype that is consistent with other models of activated tyrosine kinases over long disease latencies. Histopathological analysis of the thymi revealed effacement of normal thymic architecture as well as T cell infiltrate into the surrounding skeletal muscle. In addition, flow cytometric analysis revealed a significant increase in the CD4+/CD8+ T cell population in the thymi of these animals. No disease was observed in secondary transplant recipients following 60 days of observation. In conclusion, these results indicate that NUP214-ABL1 is able to cause either a myeloproliferative disease or a T cell lymphoma over longer latencies in mice, the latter being similar to the phenotype observed in humans with expression of the NUP214-ABL1 fusion. These findings provide a useful model for future experiments to determine if there is a contribution of other mutations together with the NUP214-ABL1 fusion towards the development of a T-ALL phenotype in mice.

Oncogenic NOTCH signaling is a major driver of T-cell transformation in T-cell acute lymphoblastic leukemia (T-ALL). However, clinical studies testing the efficacy of NOTCH1 inactivation with γ-secretase inhibitors (GSIs) have shown limited antileukemic activity for these drugs as single agents. Here we used an expression-based virtual screening approach and network perturbation analyses to identify and functionally characterize new highly active antileukemic drugs synergistic with NOTCH1 inhibition in T-ALL. Gene expression profiling studies have shown a prominent gene expression signature dominated by genes involved in growth and metabolism downstream of NOTCH1 in T-ALL. Notably, loss of the PTEN tumor suppressor gene confers resistance to GSI therapy and effectively rescues the gene expression signature induced by NOTCH1 inhibition in T-ALL. We hypothesized that drugs inducing transcriptional programs overlapping with those driven by NOTCH1 inhibition and antagonizing those resulting from PTEN loss could have synergistic antileukemic effects with GSIs in PTEN wild type and PTEN null leukemia cells. To address this question we generated gene expression signatures from Pten conditional-inducible knockout NOTCH1-driven leukemias in basal condition, upon NOTCH1 inhibition by GSI treatment and upon deletion of Pten. Connectivity Map (cMAP) analysis in this series identified 17 high scoring compounds as candidate antileukemic drugs (p To address the mechanisms mediating the antileukemic effects of withaferin A we performed a detailed analysis of the gene expression signatures induced by this drug in T-ALL lymphoblasts. These studies revealed a strong enrichment of downregulated genes involved in translation regulation in T-ALL cells upon treatment with withaferin A (p Disclosures Califano: Therasis Inc: Employment; Cancer Genetics Inc: Consultancy; Ipsen pharmaceuticals: Consultancy; Thermo Fischer Scientific: Consultancy.

The NOTCH pathway is a key regulator of developmental choices, differentiation and function throughout the hematopoietic system. An aberrant activation of this pathway may underlie leukemogenesis, as shown in T-ALL cases with the rare t(7;9)(q35;q34) targeting NOTCH1 or with mutation(s) of NOTCH1 found in approximately 50% of T-ALL cases. We report here evidence for NOTCH1 and NOTCH2 rearrangements in B-cell lymphoma. Involvement of NOTCH1 was identified by the presence of dic(9;14)(q34;q32) and trisomy 12 in a case of chronic lymphocytic leukemia (CLL) at the time of transformation, and by st(9;22)(q34;q11) accompanied by t(14;18)(q32;q21)/IGH-BCL2 and t(8;14)(q24;q32)/IGH-CMYC in a follicular lymphoma (FL). FISH analysis of dic(9;14) and t(9;22) demonstrated the respective involvement of the IGH and IGL loci, and mapped both 9q34 breakpoints to the 5′ end of NOTCH1. Western blot analysis with cleaved Notch1 (Val1744) antibody was performed in both cases and hyperactive NOTCH1 signaling was found in the first case only. This particular case displayed in addition a mutation in the PEST domain of NOTCH1. No mutations, neither in the PEST, nor in HD domain of NOTCH1 (2 domains frequently mutated in T-ALL) were found in the FL case. FISH analysis of 30 additional lymphoma cases documented by structural aberrations of 9q33q34 revealed a normal status of NOTCH1. NOTCH2 rearrangement was identified by FISH in 1 out of the 3 leukemia/lymphoma cases we could collect with t(1;14)(p13;q32). This particular CLL case showed a 3-way translocation, t(1;14;18)(p13;q32;q21), targeting also BCL2. So far, this patient did not show any clinical features of CLL transformation. We hypothesize that IG-translocations affecting NOTCH genes in B-cell malignancies contribute to the lymphomagenesis by deregulating the transcription of these genes resulting in an aberrant ligand-independent NOTCH signaling. Mutation in the PEST domain of NOTCH1 as found in one case with dic(9;14) may further contribute to the process by stabilization of the aberrant product. Further molecular and immunohistochemical investigations of these cases are in progress.