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Acute lymphoblastic leukemia (ALL) is an aggressive blood cancer that mainly affects children. Relapse rates are high and toxic chemotherapies that block DNA replication and induce DNA damage lead to health problems later in life, underlining the need for improved therapies. MYC is a transcription factor that is hyperactive in a large proportion of cancers including leukemia but is difficult to target in therapy. We show that ablation of the function of the BTB/POZ domain factor Zbtb17 (Miz-1), an important cofactor of c-Myc, significantly delayed T- and B-ALL/lymphoma in mice and interfered with the oncogenic transcriptional activity of c-Myc. Leukemic cells that still emerged in this system activated DNA replication pathways that could be targeted by current chemotherapeutic drugs such as cytarabine. Acute ablation of the Miz-1 POZ domain enhanced the effect of cytarabine treatment. The combined treatment was effective in both Eμ-Myc and Notch ICN-driven leukemia models and prolonged survival of tumor-bearing animals by accelerating apoptosis of leukemic cells. These observations suggest that targeting MIZ-1 could render current ALL chemotherapies more effective, with a better outcome for patients.Significance:Ablation of the POZ domain of Miz-1 perturbs its interaction with c-MYC and delays the generation of T- and B-cell leukemias and lymphomas.

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Supplementary Figures + Supplementary Materials and Methods

Supplementary Tables 1-2, Figure 1 from Mouse Mammary Tumor Virus p75 and p110 CUX1 Transgenic Mice Develop Mammary Tumors of Various Histologic Types

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the β-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA–mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/β-catenin autocrine loop. [Cancer Res 2009;69(18):7188–97]

GFI1 is a DNA-binding transcription factor that regulates hematopoiesis by repressing target genes through its association with complexes containing histone demethylases such as KDM1A (LSD1) and histone deacetylases (HDACs). To study the consequences of the disruption of the complex between GFI1 and histone-modifying enzymes, we have used knock-in mice harboring a P2A mutation in GFI1 coding region that renders it unable to bind LSD1 and associated histone-modifying enzymes such as HDACs. GFI1P2A mice die prematurely and show increased numbers of memory effector and regulatory T cells in the spleen accompanied by a severe systemic inflammation with high serum levels of IL-6, TNF-α, and IL-1β and overexpression of the gene encoding the cytokine oncostatin M (OSM). We identified lung alveolar macrophages, CD8 T cell from the spleen and thymic eosinophils, and monocytes as the sources of these cytokines in GFI1P2A mice. Chromatin immunoprecipitation showed that GFI1/LSD1 complexes occupy sites at the Osm promoter and an intragenic region of the Tnfα gene and that a GFI1P2A mutant still remains bound at these sites even without LSD1. Methylation and acetylation of histone H3 at these sites were enriched in cells from GFI1P2A mice, the H3K27 acetylation being the most significant. These data suggest that the histone modification facilitated by GFI1 is critical to control inflammatory pathways in different cell types, including monocytes and eosinophils, and that a disruption of GFI1-associated complexes can lead to systemic inflammation with fatal consequences.

Background Fluoropyrimidines are used in chemotherapy combinations for multiple cancers. Deficient dihydropyrimidine dehydrogenase activity can lead to severe life-threatening toxicities. DPYD*2A polymorphism is one of the most studied variants. The study objective was to document the impact of implementing this test in routine clinical practice. Methods We retrospectively performed chart reviews of all patients who tested positive for a heterozygous or homozygous DPYD*2A mutation in samples obtained from patients throughout the province of Quebec, Canada. Results During a period of 17 months, 2,617 patients were tested: 25 patients tested positive. All were White. Twenty-four of the 25 patients were heterozygous (0.92%), and one was homozygous (0.038%). Data were available for 20 patients: 15 were tested upfront, whereas five were identified after severe toxicities. Of the five patients confirmed after toxicities, all had grade 4 cytopenias, 80% grade ≥3 mucositis, 20% grade 3 rash, and 20% grade 3 diarrhea. Eight patients identified with DPYD*2A mutation prior to treatment received fluoropyrimidine-based chemotherapy at reduced initial doses. The average fluoropyrimidine dose intensity during chemotherapy was 50%. No grade ≥3 toxicities were observed. DPYD*2A test results were available in an average of 6 days, causing no significant delays in treatment initiation. Conclusion Upfront genotyping before fluoropyrimidine-based treatment is feasible in clinical practice and can prevent severe toxicities and hospitalizations without delaying treatment initiation. The administration of chemotherapy at reduced doses appears to be safe in patients heterozygous for DPYD*2A. Implications for Practice Fluoropyrimidines are part of chemotherapy combinations for multiple cancers. Deficient dihydropyrimidine dehydrogenase activity can lead to severe life-threatening toxicities. This retrospective analysis demonstrates that upfront genotyping of DPYD before fluoropyrimidine-based treatment is feasible in clinical practice and can prevent severe toxicities and hospitalizations without delaying treatment initiation. This approach was reported previously, but insufficient data concerning its application in real practice are available. This is likely the first reported experience of systematic DPYD genotyping all over Canada and North America as well.

Acute lymphoblastic leukemia (ALL) is an aggressive blood cancer that mainly affects children. Relapse rates are high and toxic chemotherapies that block DNA replication and induce DNA damage lead to health problems later in life, underlining the need for improved therapies. MYC is a transcription factor that is hyperactive in a large proportion of cancers including leukemia but is difficult to target in therapy. We show that ablation of the function of the BTB/POZ domain factor Zbtb17 (Miz-1), an important cofactor of c-Myc, significantly delayed T- and B-ALL/lymphoma in mice and interfered with the oncogenic transcriptional activity of c-Myc. Leukemic cells that still emerged in this system activated DNA replication pathways that could be targeted by current chemotherapeutic drugs such as cytarabine. Acute ablation of the Miz-1 POZ domain enhanced the effect of cytarabine treatment. The combined treatment was effective in both Eμ-Myc and Notch ICN-driven leukemia models and prolonged survival of tumor-bearing animals by accelerating apoptosis of leukemic cells. These observations suggest that targeting MIZ-1 could render current ALL chemotherapies more effective, with a better outcome for patients. Significance: Ablation of the POZ domain of Miz-1 perturbs its interaction with c-MYC and delays the generation of T- and B-cell leukemias and lymphomas.

GFI1 is a DNA-binding transcription factor that regulates hematopoiesis by repressing target genes through its association with complexes containing histone demethylases such as KDM1A (LSD1) and histone deacetylases (HDACs). To study the consequences of the disruption of the complex between GFI1 and histone-modifying enzymes, we have used knock-in mice harboring a P2A mutation in GFI1 coding region that renders it unable to bind LSD1 and associated histone-modifying enzymes such as HDACs. GFI1

Here we demonstrate a mode of reciprocal regulation between GFI1 and p53 that controls the induction of apoptosis in T cells. We show that GFI1 prevents induction of p53 dependent apoptosis by recruiting LSD1 to p53, which leads to the demethylation of its C-terminal domain. This is accompanied by a decrease of the acetylation of lysine 117 within the core domain of the murine p53 protein, which is required for transcriptional induction of apoptosis. Our results support a model in which the effect of GFI1’s regulation of methylation at the c-terminus of p53 is ultimately mediated through control of acetylation at lysine 117 of p53. We propose that GFI1 acts prior to the occurrence of DNA damage to affect the post-translational modification state and limit the subsequent activation of p53. Once activated, p53 then transcriptionally activates GFI1, presumably in order to re-establish the homeostatic balance of p53 activity. These findings have implications for the activity level of p53 in various disease contexts where levels of GFI1 are either increased or decreased.

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with β-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/β-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite β-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/β-catenin target genes. Consistently, numerous canonical Wnt/β-catenin target genes, co-occupied by Gfi1b, β-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/β-catenin signaling pathway., Gfi1b regulates cellularity of haematopoietic stem cells (HSCs) and megakaryocytes (MKs) as well as spreading of MKs on matrix. Here the authors show that Gfi1b regulates this behaviour by recruiting LSD1 and β-catenin to Wnt/β-catenin signalling targets.

Mutations in GFI1B are associated with inherited bleeding disorders called GFI1B-related thrombocytopenias. We show here that mice with a megakaryocyte-specific Gfi1b deletion exhibit a macrothrombocytopenic phenotype along a megakaryocytic dysplasia reminiscent of GFI1B-related thrombocytopenia. GFI1B deficiency increases megakaryocyte proliferation and affects their ploidy, but also abrogates their responsiveness towards integrin signaling and their ability to spread and reorganize their cytoskeleton. Gfi1b-null megakaryocytes are also unable to form proplatelets, a process independent of integrin signaling. GFI1B-deficient megakaryocytes exhibit aberrant expression of several components of both the actin and microtubule cytoskeleton, with a dramatic reduction of α-tubulin. Inhibition of FAK or ROCK, both important for actin cytoskeleton organization and integrin signaling, only partially restored their response to integrin ligands, but the inhibition of PAK, a regulator of the actin cytoskeleton, completely rescued the responsiveness of Gfi1b-null megakaryocytes to ligands, but not their ability to form proplatelets. We conclude that Gfi1b controls major functions of megakaryocytes such as integrin-dependent cytoskeleton organization, spreading and migration through the regulation of PAK activity whereas the proplatelet formation defect in GFI1B-deficient megakaryocytes is due, at least partially, to an insufficient α-tubulin content.

6580 Background: The combination of carboplatin and 5-fluorouracil (5-FU) is effective when used concurrently with radiotherapy for locoregionally advanced oropharyngeal carcinomas (Calais et al. 1999). DPYD polymorphisms can be associated with an increased risk of severe toxicity to fluoropyrimidines (Deenen et al. 2016). Upfront screening for the DPYD*2A allele is available in the province of Québec, Canada since March 2017. This study aimed to determine the effect of upfront genotyping on grade ≥3 toxicities. Methods: The studied population included all consecutive cases of oropharyngeal carcinomas treated with 5-FU based chemoradiotherapy one year before and after the implementation of upfront DPYD*2A genotyping. All patients were treated at the Centre Hospitalier de l’Université de Montréal (CHUM) between March 2016 and April 2018. Clinical data were extracted from chart review. Extended screening for 3 supplemental at-risk DPYD variants was also retrospectively performed in August 2019. Results: 181 patients were included in the analysis (87 patients before and 94 patients after DPYD*2A screening implementation). 91% of patients (n = 86) were prospectively genotyped for the DPYD*2A allele. Of those screened, 2% (n = 2/87) demonstrated a heterozygous DPYD*2A mutation. Those two patients received cisplatin-based treatment and thus avoided 5-FU toxicities. Extended genotyping of DPYD*2A-negative patients later allowed for the retrospective identification of 6 additional patients with alternative DPYD variants (two c.2846A > T and four c.1236G > A allele mutations). Conclusions: The DPYD*2A, c.2846A > T and c.1236G > A polymorphisms are associated with an increased risk of G3-4 toxicity to 5-FU, as well as higher hospitalization rates. Upfront DPYD genotyping can identify patients in whom fluoropyrimidine-related toxicity should be avoided. This represents an interesting addition in terms of pharmacovigilance. [Table: see text]

Growth factor independent 1 (Gfi1) controls myeloid differentiation by regulating gene expression and limits the activation of p53 by facilitating its de-methylation at Lysine 372. In human myeloid leukemia, low GFI1 levels correlate with an inferior prognosis. Here, we show that knockdown (KD) of Gfi1 in mice causes a fatal myeloproliferative disease (MPN) that could progress to leukemia after additional mutations. Both KO and KD mice accumulate myeloid cells that show signs of metabolic stress and high levels of reactive oxygen species. However, only KO cells have elevated levels of Lysine 372 methylated p53. This suggests that in contrast to absence of GFI1, KD of GFI1 leads to the accumulation of myeloid cells because sufficient amount of GFI1 is present to impede p53-mediated cell death, leading to a fatal MPN. The combination of myeloid accumulation and the ability to counteract p53 activity under metabolic stress could explain the role of reduced GF1 expression in human myeloid leukemia.

// Zubaidah M. Ramdzan 1 , Ranjana Pal 1, 5 , Simran Kaur 1, 2 , Lam Leduy 1 , Ginette Berube 1 , Sayeh Davoudi 1, 2 , Charles Vadnais 1, 2 , Alain Nepveu 1, 2, 3, 4 1 Goodman Cancer Centre, McGill University, Montreal, Quebec H3A 1A3, Canada 2 Department of Biochemistry, McGill University, Montreal, Quebec H3A 1A3, Canada 3 Department of Medicine, McGill University, Montreal, Quebec H3A 1A3, Canada 4 Department of Oncology, McGill University, Montreal, Quebec H3A 1A3, Canada 5 Department of Biological Sciences, Presidency University, Kolkata 700073, India Correspondence to: Alain Nepveu, e-mail: alain.nepveu@mcgill.ca Keywords: cellular senescence, mouse embryo fibroblasts, oxidative DNA damage, 8-oxoguanine DNA glycosylase 1 (OGG1), CUX1 Received: November 17, 2014 Accepted: December 11, 2014 Published: January 08, 2015 ABSTRACT Despite having long telomeres, mouse embryo fibroblasts (MEFs) senesce more rapidly than human diploid fibroblasts because of the accumulation of oxidative DNA damage. The CUX1 homeodomain protein was recently found to prevent senescence in RAS-driven cancer cells that produce elevated levels of reactive-oxygen species. Here we show that Cux1 −/− MEFs are unable to proliferate in atmospheric (20%) oxygen although they can proliferate normally in physiological (3%) oxygen levels. CUX1 contains three domains called Cut repeats. Structure/function analysis established that a single Cut repeat domain can stimulate the DNA binding, Schiff-base formation, glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1, OGG1. Strikingly and in contrast to previous reports, OGG1 exhibits efficient AP-lyase activity in the presence of a Cut repeat. Repair of oxidative DNA damage and proliferation in 20% oxygen were both rescued in Cux1 −/− MEFs by ectopic expression of CUX1 or of a recombinant Cut repeat protein that stimulates OGG1 but is devoid of transcription activation potential. These findings reinforce the causal link between oxidative DNA damage and cellular senescence and suggest that the role of CUX1 as an accessory factor in DNA repair will be critical in physiological situations that generate higher levels of reactive oxygen species.

GFI1 is a transcriptional regulator expressed in lymphoid cells, and an “oncorequisite” factor required for development and maintenance of T-lymphoid leukemia. GFI1 deletion causes hypersensitivity to ionizing radiation, for which the molecular mechanism remains unknown. Here, we demonstrate that GFI1 is required in T cells for the regulation of key DNA damage signaling and repair proteins. Specifically, GFI1 interacts with the arginine methyltransferase PRMT1 and its substrates MRE11 and 53BP1. We demonstrate that GFI1 enables PRMT1 to bind and methylate MRE11 and 53BP1, which is necessary for their function in the DNA damage response. Thus, our results provide evidence that GFI1 can adopt non-transcriptional roles, mediating the post-translational modification of proteins involved in DNA repair. These findings have direct implications for treatment responses in tumors overexpressing GFI1 and suggest that GFI1’s activity may be a therapeutic target in these malignancies., The transcription factor GFI1 mediates the DNA damage response (DDR) of T cells through a yet unknown mechanism. Here the authors show that GFI1 can adopt non-transcriptional roles during DDR, enabling PRMT1 to bind and methylate the DNA repair proteins MRE11 and 53BP1.

The p110 Cut homeobox 1 (CUX1) transcription factor regulates genes involved in DNA replication and chromosome segregation. Using a genome-wide-approach, we now demonstrate that CUX1 also modulates the constitutive expression of DNA damage response genes, including ones encoding ATM and ATR, as well as proteins involved in DNA damage-induced activation of, and signaling through, these kinases. Consistently, RNAi knockdown or genetic inactivation of CUX1 reduced ATM/ATR expression and negatively impacted hallmark protective responses mediated by ATM and ATR following exposure to ionizing radiation (IR) and UV, respectively. Specifically, abrogation of CUX1 strongly reduced ATM autophosphorylation after IR, in turn causing substantial decreases in (i) levels of phospho-Chk2 and p53, (ii) γ-H2AX and Rad51 DNA damage foci and (iii) the efficiency of DNA strand break repair. Similarly remarkable reductions in ATR-dependent responses, including phosphorylation of Chk1 and H2AX, were observed post-UV. Finally, multiple cell cycle checkpoints and clonogenic survival were compromised in CUX1 knockdown cells. Our results indicate that CUX1 regulates a transcriptional program that is necessary to mount an efficient response to mutagenic insult. Thus, CUX1 ensures not only the proper duplication and segregation of the genetic material, but also the preservation of its integrity.

Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2 + subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome.

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the β-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA–mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/β-catenin autocrine loop. [Cancer Res 2009;69(18):7188–97]

The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase alpha gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase alpha gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage.

The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.

Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).

Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting histone deacetylases to target genes. We present data that low expression of GFI1 is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1 expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. Leukemic cells from animals that express low levels of GFI1 show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1 expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KD mice and from AML patients with low GFI1 levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1 expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.

The base excision repair (BER) that repairs oxidative damage is upregulated as an adaptive response in maintaining tumorigenesis of RAS-transformed cancer cells., The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1+/− MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between KrasG12V and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway., Author Summary In the context of tumor development and progression, mutations are believed to accumulate owing to compromised DNA repair. Such mutations promote oncogenic growth. Yet cancer cells also need to sustain a certain level of DNA repair in order to replicate their DNA and successfully proliferate. Here we show that cancer cells that harbor an activated RAS oncogene exhibit heightened DNA repair capability, specifically in the base excision repair (BER) pathway that repairs oxidative DNA damage. RAS oncogenes alone do not transform primary cells but rather cause their senescence—that is, they stop dividing. As such, cellular senescence in this context is proposed to function as a tumor-suppressive mechanism. We show that CUX1, a protein that accelerates oxidative DNA damage repair, prevents cells from senescing and enables proliferation in the presence of a RAS oncogene. Consistent with this, RAS-induced senescence is also prevented by ectopic expression of OGG1, the DNA glycosylase that removes 8-oxoguanine, the most abundant oxidized base. Strikingly, CUX1 expression in transgenic mice enables the emergence of tumors with spontaneous activating Kras mutations. Conversely, knockdown of CUX1 is synthetic lethal for RAS-transformed cells, thereby revealing a potential Achilles' heel of these cancer cells. Overall, the work provides insight into understanding the role of DNA repair in cancer progression, showing that while DNA damage-induced mutations promote tumorigenesis, sustained RAS-dependent tumorigenesis requires suppression of DNA damage. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.

Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36S)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells in vitro and in vivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted. OA gold - CA Khandanpour

Background Overexpression of the Cut homeobox 1 gene, CUX1, inverselycorrelates with patient survival in breast cancers. Cell-based assays andmolecular studies have revealed that transcriptional regulation byCUX1 involves mostly the proteolytically processed p110isoform. As there is no antibody specific to p110 CUX1 only, an alternatestrategy must be employed to identify its targets. Results We expressed physiological levels of a tagged-p110 CUX1 protein and performedchromatin affinity purification followed by hybridization on ENCODE andpromoter arrays. Targets were validated by chromatin immunoprecipitation andtranscriptional regulation by CUX1 was analyzed in expression profiling andRT-qPCR assays following CUX1 knockdown or p110 CUX1 overexpression.Approximately 47% and 14% of CUX1 binding sites were respectively mappedless than 4 Kbp, or more than 40 Kbp, away from a transcription start site.More genes exhibited changes in expression following CUX1 knockdown thanp110 CUX1 overexpression. CUX1 directly activated or repressed 7.4% and 8.4%of putative targets identified on the ENCODE and promoter arraysrespectively. This proportion increased to 11.2% for targets with 2 bindingsites or more. Transcriptional repression was observed in a slightly higherproportion of target genes. The CUX1 consensus binding motif, ATCRAT, wasfound at 47.2% of the CUX1 binding sites, yet only 8.3% of the CUX1consensus motifs present on the array were bound in vivo. Thepresence of a consensus binding motif did not have an impact on whether atarget gene was repressed or activated. Interestingly, the distance betweena binding site and a transcription start site did not significantly reducedthe ability of CUX1 to regulate a target gene. Moreover, CUX1 not only wasable to regulate the next adjacent gene, but also regulated the gene locatedbeyond this one as well as the gene located further away in the oppositedirection. Conclusion Our results demonstrate that p110 CUX1 can activate or repress transcriptionwhen bound at a distance and can regulate more than one gene on certaingenomic loci.

Nature Communications 2: Article number: 338 (2011); Published: 7 June 2012; Updated: 25 September 2012. This Article contains errors in the numbering of several papers in the reference list; reference 34 is incorrectly listed as reference 44 and references 35 to 44 are incorrectly listed as 34 to 43.

α-fetoprotein (AFP) is not only a widely used biomarker in hepatocellular carcinoma (HCC) surveillance, but is also clinically recognized as linked with aggressive tumour behaviour. Here we show that deregulation of microRNA122, a liver-specific microRNA, is a cause of both AFP elevation and a more biologically aggressive phenotype in HCC. We identify CUX1, a direct target of microRNA122, as a common central mediator of these two effects. Using liver tissues from transgenic mice in which microRNA122 is functionally silenced, an orthotopic xenograft tumour model, and human clinical samples, we further demonstrate that a microRNA122/CUX1/microRNA214/ZBTB20 pathway regulates AFP expression. We also show that the microRNA122/CUX1/RhoA pathway regulates the aggressive characteristics of tumours. We conclude that microRNA122 and associated signalling proteins may represent viable therapeutic targets, and that serum AFP levels in HCC patients may be a surrogate marker for deregulated intracellular microRNA122 signalling pathways in HCC tissues.

Background Lats1 (large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation. Results Genome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1. Conclusion While inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of Lats1.

In this study, we investigated the mechanism by which the CUX1 transcription factor can stimulate cell migration and invasion. The full-length p200 CUX1 had a weaker effect than the proteolytically processed p110 isoform; moreover, treatments that affect processing similarly impacted cell migration. We conclude that the stimulatory effect of p200 CUX1 is mediated in part, if not entirely, through the generation of p110 CUX1. We established a list of putative transcriptional targets with functions related to cell motility, and we then identified those targets whose expression was directly regulated by CUX1 in a cell line whose migratory potential was strongly stimulated by CUX1. We identified 18 genes whose expression was directly modulated by p110 CUX1, and its binding to all target promoters was validated in independent chromatin immunoprecipitation assays. These genes code for regulators of Rho-GTPases, cell-cell and cell-matrix adhesion proteins, cytoskeleton-associated proteins, and markers of epithelial-to-mesenchymal transition. Interestingly, p110 CUX1 activated the expression of genes that promote cell motility and at the same time repressed genes that inhibit this process. Therefore, the role of p110 CUX1 in cell motility involves its functions in both activation and repression of transcription. This was best exemplified in the regulation of the E-cadherin gene. Indeed, we uncovered a regulatory cascade whereby p110 CUX1 binds to the snail and slug gene promoters, activates their expression, and then cooperates with these transcription factors in the repression of the E-cadherin gene, thereby causing disorganization of cell-cell junctions.

Objective: The Myc-interacting zinc finger protein 1 (Miz-1) is a ubiquitous BTB/POZ domain and zinc finger protein that acts as both a transcriptional repressor and activator. The POZ domain of Miz-1 enables protein-protein interactions and facilitates a stable association with chromatin. Miz-1 binds to the proto-oncogen c-Myc and modulates c-Myc target gene expression. Overexpression of c-Myc is an important feature of Burkitt-type B cell lymphomas (BL) caused by c-Myc gene rearrangements. The aim of this project is to determine if Miz-1 is a crucial collaborator of c-Myc in transcriptional regulation of B cells and in the development of c-Myc-dependent B-cell lymphomas. Methods: We are using the Eµ-Myc mouse model to generate Burkitt type c-Myc dependent lymphomas to study the implication of Miz-1 in a c-Myc driven process of malignant transformation. Mice carrying the Eµ-Myc transgene (c-Myc gene placed under the control of Eµ enhancer of the IgH locus) overexpress c-Myc in lymphoid cells and develop a disease similar to Burkitt's lymphoma. Mice that express a conditional non-functional Miz-1 allele lacking the part coding for the POZ domain in B cells (Mb1-Cre-Miz-1fl/fl mice, hereafter called ΔPOZ mice) were crossed with Eµ-Myc transgenic mice. We monitored Eµ-Myc animals expressing wild type or ΔPOZ Miz-1 protein over a period of 500 days. Incidence and latency periods of the development of tumors in both cohorts was compared. Pre-cancerous mice of all genotypes were also investigated to evaluate the influence of Miz-1 during the development of the c-Myc dependant lymphomas. To identify underlying molecular mechanisms, global mRNA expression profiles of pre-neoplastic and tumor cells derived from Eµ-Myc and ΔPOZ/Eµ-Myc mice were performed using high throughput sequencing (RNA-seq). To address the role of Miz-1 in the deregulation of chromatin associated with c-Myc overexpression, chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) was performed on B-lymphoma and pre-tumoral B-cells and compared to normal B-cells. Binding to chromatin of Miz-1 and c-Myc was assessed. Results: ΔPOZ/Eµ-Myc mice develop lymphomas with a significant longer latency and lower incidence than Eµ-Myc mice expressing functional Miz-1 proteins. Additionally, blood analysis of sick animals reveal a lower amount of Large Unstained Cells (LUC) when Miz-1 is mutated. Accordingly, in 40 day old pre-tumoral ΔPOZ/Eµ-Myc animals compared to Eµ-Myc mice, less pre-B and/or pro-B cells were observed in lymphoid organs and little or no LUC were present in the blood. This suggests that Myc driven lymphoma and leukemia is impaired when a ΔPOZ Miz-1 protein is expressed. Our RNA-seq analyses of pre-neoplastic and tumor cells revealed that the expression of ΔPOZ Miz-1 induces deregulation of several genes belonging to different GO (Gene Ontology) functions like “small GTPase-mediated signal transduction”. Interestingly, comparision of RNA-seq and Miz-1 ChIP-seq analyses indicates that some Miz-1 bound genes are deregulated in cells from ΔPOZ/Eµ-Myc mice (e.g: Aurora kinase A (Aurka)). Conclusion: Our data indicate that Miz-1 is required for the efficient development of aggressive c-Myc-driven lymphomas. Miz-1 is probably cooperating with c-Myc in the abnormal transcriptional program that is occuring in lymphoma cells. Therefore, targeting Miz-1 in B-lymphomas could lead to the development of new therapeutic approaches. Citation Format: Julie Ross, René Winkler, Charles Vadnais, Marissa Rashkovan, Christian Kosan, Tarik Möröy. Loss of functional Miz-1 impairs c-Myc-dependent B cell lymphomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B08.

The Myc-Interacting Zinc-finger protein-1 (Miz-1) can bind to the proto-oncogene c-Myc and the Miz-1/c-Myc complex can regulate the transcription of specific target genes. Miz-1 contains a BTB/POZ domain and thirteen zinc finger domains that mediate binding to DNA and its interaction with partner proteins. While Miz-1 itself functions as a transcriptional transactivator, it enables c-Myc to act as a transcriptional repressor of genes negatively regulating cell cycle progression. We have previously reported that Miz-1 plays an essential role during early stages of T cell development by regulating the translation of p53 via the ribosomal protein L22. Based on this function of Miz-1 in T cell development, we have decided to investigate a potential role of Miz-1 in the development of T cell leukemia or lymphoma. We have used several different experimental models to induce T-ALL in mice that are conditionally deficient for Miz-1. We used non-acute transforming retroviruses, such as Moloney Murine Leukemia Virus (MMLV), which induces T-cell leukemia by activating known oncogenes, including Gfi1, Pim1 and c-Myc via proviral insertion. We also used ENU mutagenesis, which induces T-cell leukemia through activation of the Ras pathway. We observed that Miz-1 is required for the development of T cell leukemia regardless of whether it is induced by MMLV or ENU, since Miz-1-deficient mice succumb to the disease with a significantly longer latency period than WT animals. Importantly, during tumorigenesis a strong counter selection occurs against the loss of Miz-1. Interestingly, MycV394D transgenic animals, carrying a c-Myc protein that can no longer interact directly with Miz-1, develop tumors at a rate comparable to WT mice, indicating that a function of Miz-1 is required for T cell tumorigenesis that is independent of its interaction with c-Myc. Transcriptome analyses showed that Miz-1 deficiency correlated with a decrease of expression in autophagy-related genes, consistent with earlier reports that Miz-1 is necessary for the induction of this pathway. ChIP-seq confirmed that Miz-1 binds to the promoters of these autophagy-related genes, indicating that these genes are activated in the presence of Miz-1, also consistent with previous reports. Furthermore, Miz-1-deficient tumors undergo higher levels of apoptosis than WT tumors. Based on these results, we conclude that Miz-1 is essential for the efficient development of T-ALL and that loss of Miz-1 significantly hinders T-ALL development in part through the inability of these cells to induce autophagy. Citation Format: Marissa Rashkovan, Charles Vadnais, Julie Ross, Jennifer Fraszczak, Tarik Moroy. Loss of Miz-1 increases latency of T-ALL by preventing induction of autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 997. doi:10.1158/1538-7445.AM2015-997

Myelodysplastic syndromes (MDS) are characterized by disturbances in the development of different blood lineages, which can progress to AML. The knowledge about the factors predisposing the development of AML is sparse. Growth factor independence 1 is a transcription factor regulating the differentiation of myeloid cells. Previously a Single Nucleotid Polymorphism (SNP) of Gfi1, denominated Gfi136N, had been described. This variant is characterized by a SNP leading to the replacement of Serine at position 36 to an Asparagine. This SNP is found in 5-7% of all Caucasians and in 10-15% of all AML patients. We sought to investigate whether Gfi136N could be a novel predictive marker for the development of AML in MDS patients. To this end, we characterized 201 patients with regard to presence of the Gfi136N variant. Patients were recruited from different German centers treating MDS patients, i.e. Essen, Düsseldorf and Dresden. Within this cohort, about 10% were heterozygous for Gfi136N. There was no difference between MDS patients heterozygous for Gfi136N or homozygous for Gfi136S with regard to age, sex, cytogenetic or IPSS score. Presence of Gfi136N significantly increased incidence (Odds ratio 2 fold) and shortened latency to AML progression (around 2 years for Gfi136N heterozygous patients compared to around 6 years for Gfi136S homozygous patients (p=0.001). To further investigate the role of Gfi136N in AML development, we generated mice expressing either the wildtype form of human Gfi1 (Gfi136S) or Gfi136N. We mated these mice with mice expressing the Nup98HoxD13 transgene. Mice expressing Nup98HoxD13 develop a MDS like disease and about 20-30% progress to AML. Mice with Nup98HoxD13 and Gfi136N alleles (n=10) developed AML with a higher incidence (60% compared to 20%) and shorter latency (200 days compared to 340 days) than mice with Nup98HoxD13 and Gfi136S alleles. (n=8, p=0.05) To confirm our finding, we used additional murine AML models resembling human AML cells. MLL-AF9 and AML1-ETO9a are recurrent so called oncofusionproteins, which are charateristic for different human AML subtypes. By retroviral transduction of murine Lineage negative (Lin neg) cells, murine leukemia resembling human AML can be generated. We transduced Lin neg cells from Gfi136S and Gfi136N mice with retroviruses expressing the onocfusionporteins MLL-AF9 or AML1-ETO9a. After seeding in semi-solid medium, transduced Gfi136N cells generated more colonies with a higher cell number than transduced Gfi136S cells (2-4 fold more cells or colonies, depending on the oncofusionprotein, p=0.05). In summary, our data suggest that Gfi136N is a novel predictive marker for AML development among MDS patients which can be recapitulated in mice. To investigate the reason behind this observation, we analyzed lineage negative ckit pos blood cells from Gfi136N or Gfi136S homozygous mice. Genome-wide analysis of histone modification showed that mice expressing the 36N variant display globally higher levels of diMeH3K4 and AcH3K9 activation marks with a significant positive correlation between them. We analyzed the genes, which had a higher level of activation marks in Gfi136N cells compared to Gfi136S. We found that pathways involved in cytokine signaling, hematopoietic lineage development and AML genesis were overrepresented among these genes. Thus we show that Gfi136N might play a crucial role in AML development of MDS patients by inducing epigenetic changes, which promote AML development. Disclosures: Germing: Celgene: Honoraria, Research Funding; Jansen-Cilag: Honoraria; Novartis: Research Funding; GSK: Research Funding; Amgen: Research Funding. Platzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.