Esther Tijchon

BTG1 regulates glucocorticoid receptor autoinduction in acute lymphoblastic leukemia

Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

B-lineage transcription factors and cooperating gene lesions required for leukemia development.

Differentiation of hematopoietic stem cells into B lymphocytes requires the concerted action of specific transcription factors, such as RUNX1, IKZF1, E2A, EBF1 and PAX5. As key determinants of normal B-cell development, B-lineage transcription factors are frequently deregulated in hematological malignancies, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and affected by either chromosomal translocations, gene deletions or point mutations. However, genetic aberrations in this developmental pathway are generally insufficient to induce BCP-ALL, and often complemented by genetic defects in cytokine receptors and tyrosine kinases (IL-7Rα, CRLF2, JAK2 and c-ABL1), transcriptional cofactors (TBL1XR1, CBP and BTG1), as well as the regulatory pathways that mediate cell-cycle control (pRB and INK4A/B). Here we provide a detailed overview of the genetic pathways that interact with these B-lineage specification factors, and describe how mutations affecting these master regulators together with cooperating lesions drive leukemia development.

The tumour antigen PRAME is a subunit of a Cul2 ubiquitin ligase and associates with active NFY promoters

The human tumour antigen PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in tumours. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumourigenesis remain largely elusive. We applied protein‐complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2‐based E3 ubiquitin ligase. PRAME can be recruited to DNA in vitro, and genome‐wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY and at enhancers. Our results are consistent with a role for the PRAME ubiquitin ligase complex in NFY‐mediated transcriptional regulation.

The human EKC/KEOPS complex is recruited to Cullin2 ubiquitin ligases by the human tumour antigen PRAME.

The human tumour antigen PRAME (preferentially expressed antigen in melanoma) is frequently overexpressed during oncogenesis, and high PRAME levels are associated with poor clinical outcome in a variety of cancers. However, the molecular pathways in which PRAME is implicated are not well understood. We recently characterized PRAME as a BC-box subunit of a Cullin2-based E3 ubiquitin ligase. In this study, we mined the PRAME interactome to a deeper level and identified specific interactions with OSGEP and LAGE3, which are human orthologues of the ancient EKC/KEOPS complex. By characterizing biochemically the human EKC complex and its interactions with PRAME, we show that PRAME recruits a Cul2 ubiquitin ligase to EKC. Moreover, EKC subunits associate with PRAME target sites on chromatin. Our data reveal a novel link between the oncoprotein PRAME and the conserved EKC complex and support a role for both complexes in the same pathways.

Tumor suppressors BTG1 and IKZF1 cooperate during mouse leukemia development and increase relapse risk in B-cell precursor acute lymphoblastic leukemia patients

Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia (P=0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival (P=0.0003) and a higher 5-year cumulative incidence of relapse (P=0.005), when compared with IKZF1-deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1, did not affect the outcome of IKZF1-deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1-deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1+/− mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1+/− displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function.

Tumor suppressors BTG1 and BTG2 regulate early mouse B-cell development

Transcription factors that regulate B-cell differentiation are frequently targeted by genetic aberrations in B-cell malignancies. The B-cell translocation gene 1 ( BTG1 ), which encodes a transcription coregulator,[1][1],[2][2] is recurrently affected by gene deletions in B-cell precursor acute

The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.

Targeted Deletion of Btg1 and Btg2 Results in Homeotic Transformation of the Axial Skeleton

Btg1 and Btg2 encode highly homologous proteins that are broadly expressed in different cell lineages, and have been implicated in different types of cancer. Btg1 and Btg2 have been shown to modulate the function of different transcriptional regulators, including Hox and Smad transcription factors. In this study, we examined the in vivo role of the mouse Btg1 and Btg2 genes in specifying the regional identity of the axial skeleton. Therefore, we examined the phenotype of Btg1 and Btg2 single knockout mice, as well as novel generated Btg1 -/-;Btg2 -/- double knockout mice, which were viable, but displayed a non-mendelian inheritance and smaller litter size. We observed both unique and overlapping phenotypes reminiscent of homeotic transformation along the anterior-posterior axis in the single and combined Btg1 and Btg2 knockout animals. Both Btg1 -/- and Btg2 -/- mice displayed partial posterior transformation of the seventh cervical vertebra, which was more pronounced in Btg1 -/-;Btg2 -/- mice, demonstrating that Btg1 and Btg2 act in synergy. Loss of Btg2, but not Btg1, was sufficient for complete posterior transformation of the thirteenth thoracic vertebra to the first lumbar vertebra. Moreover, Btg2 -/- animals displayed complete posterior transformation of the sixth lumbar vertebra to the first sacral vertebra, which was only partially present at a low frequency in Btg1 -/- mice. The Btg1 -/-;Btg2 -/- animals showed an even stronger phenotype, with L5 to S1 transformation. Together, these data show that both Btg1 and Btg2 are required for normal vertebral patterning of the axial skeleton, but each gene contributes differently in specifying the identity along the anterior-posterior axis of the skeleton.

Tumor suppressor BTG1 promotes PRMT1-mediated ATF4 function in response to cellular stress

Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses.

CBFβ-MYH11 interferes with megakaryocyte differentiation via modulating a gene program that includes GATA2 and KLF1

The inv(16) acute myeloid leukemia associated CBFβ-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFβ - MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFβ-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1 binding sites upon fusion protein knockdown, and overexpression of GATA2 partly restores megakaryocyte directed differentiation suppressed by CBFβ-MYH11. Together, our findings suggest that in inv(16) leukemia, the CBFβ-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.