PBX3 and MEIS1 Cooperate in Hematopoietic Cells to Drive Acute Myeloid Leukemias Characterized by a Core Transcriptome of the MLL-Rearranged Disease

Overexpression of HOXA/MEIS1/PBX3 homeobox genes is the hallmark of mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). HOXA9 and MEIS1 are considered to be the most critical targets of MLL fusions and their co-expression rapidly induces AML. MEIS1 and PBX3 are not individually able to transform cells and were therefore hypothesized to function as cofactors of HOXA9. However, in this study we demonstrate that co-expression of PBX3 and MEIS1 (PBX3/MEIS1), without ectopic expression of a HOX gene, is sufficient for transformation of normal mouse hematopoietic stem/progenitor cells in vitro. Moreover, PBX3/MEIS1 overexpression also caused AML in vivo, with a leukemic latency similar to that caused by forced expression of MLL-AF9, the most common form of MLL fusions. Furthermore, gene expression profiling of hematopoietic cells demonstrated that PBX3/MEIS1 overexpression, but not HOXA9/MEIS1, HOXA9/PBX3 or HOXA9 overexpression, recapitulated the MLL-fusion-mediated core transcriptome, particularly upregulation of the endogenous Hoxa genes. Disruption of the binding between MEIS1 and PBX3 diminished PBX3/MEIS1-mediated cell transformation and HOX gene upregulation. Collectively, our studies strongly implicate the PBX3/MEIS1 interaction as a driver of cell transformation and leukemogenesis, and suggest that this axis may play a critical role in the regulation of the core transcriptional programs activated in MLL-rearranged and HOX-overexpressing AML. Therefore, targeting the MEIS1/PBX3 interaction may represent a promising therapeutic strategy to treat these AML subtypes

miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy

Deciphering How Genomic Instability in Hematopoietic Cells Drives Tumorigenesis

Mutations in DNA repair factors are increasingly recognized for having deleterious effects on hematopoiesis and increasing risk for developing hematopoietic malignancies (HMs). These genomically unstable cancers exhibit increases in somatic mutation rates as well as cases of large-scale chromosomal aberrations and translocations. In my thesis work, I investigated alterations in the homologous recombination (HR) DNA repair pathway, centered on the ATM-CHEK2-BRCA1 response to double strand breaks (DSBs). My work addresses the mechanisms of genomic instability that leads to an HM due to alterations in HR repair and effects on the cellular response to DNA damage and replication stress. The findings from my thesis work highlight the dual role that HR-associated factors play at stressed replication forks and implicate replication-mediated DNA damage in the etiology of genomic instability in hematopoietic cells. I investigated the mechanisms of genomic instability leading to recurrent Tcra/Myc-Pvt1 translocations in a T-cell leukemia model with aberrantly stabilized β-catenin. I show that DSBs in the Tcra site of the translocation are Rag-generated whereas the Myc-Pvt1 DSBs are not. I find that aberrant activation of β-catenin in thymocytes leads to a Tcf-1 mediated downregulation of HR-pathway member that promotes the retention of replication-mediated DSBs, providing the conditions for translocations to form. I also investigated a mouse model with hematopoietic-specific knockout (KO) of Brca1 that produces bone marrow failure and HMs with widespread chromosomal aberrations. My investigations into the mechanism of this genomic instability in the absence of this central HR factor implicate replication fork restart failures and the use of more error prone backup pathway for DSB repair, including non-homologous end joining (NHEJ) and alternative end joining (alt-EJ). Importantly, I show that bone marrow that is heterozygous for Brca1 also shows mild deficiencies at stressed replication forks and increased expression of NHEJ and alt-EJ factors. Finally, I investigated germline variants in the cell cycle regulator, CHEK2, for their contribution to increased risk for developing an HM. My work helps to identify two CHEK2 alleles, c.470C>T/p.I200T and c.1283C>T/p.S428P, as increasing the odds of developing an HM for patient carriers. Furthermore, I use a mouse model of the CHEK2 p.I200T allele and show that these mice develop leukocytosis, clonal hematopoiesis, and HMs at late stages. This suggest that variants in CHEK2 can alter the proliferation rates and somatic mutation rates in hematopoietic cells, contributing to genomic instability and outgrowth of bone marrow clones. Taken together, my studies highlight the dual role that HR factors play in repairing DSBs and in managing replication stress. I show how altered function can lead to failures of both replication fork protection and DSB repair, which act synergistically to increase genomic instability in these cells. These findings contribute additional context both to our understanding of current risks for carrying a mutation in these genome maintenance genes, and also opening up new therapeutic targets for treatment of HR-deficient HMs

Tcf-1 promotes genomic instability and T cell transformation in response to aberrant β-catenin activation

Understanding the mechanisms promoting chromosomal translocations of the rearranging receptor loci in leukemia and lymphoma remains incomplete. Here we show that leukemias induced by aberrant activation of β-catenin in thymocytes, which bear recurrent Tcra/Myc-Pvt1 translocations, depend on Tcf-1. The DNA double strand breaks (DSBs) in the Tcra site of the translocation are Rag-generated, whereas the Myc-Pvt1 DSBs are not. Aberrantly activated β-catenin redirects Tcf-1 binding to novel DNA sites to alter chromatin accessibility and down-regulate genome-stability pathways. Impaired homologous recombination (HR) DNA repair and replication checkpoints lead to retention of DSBs that promote translocations and transformation of double-positive (DP) thymocytes. The resulting lymphomas, which resemble human T cell acute lymphoblastic leukemia (T-ALL), are sensitive to PARP inhibitors (PARPis). Our findings indicate that aberrant β-catenin signaling contributes to translocations in thymocytes by guiding Tcf-1 to promote the generation and retention of replication-induced DSBs allowing their coexistence with Rag-generated DSBs. Thus, PARPis could offer therapeutic options in hematologic malignancies with active Wnt/β-catenin signaling

Characterization of CpG sites that escape methylation on the inactive human X-chromosome

<p>In many whole genome studies of gene expression or modified cytosines, data from probes localized to the X-chromosome are removed from analyses due to gender bias. Previously, we observed population differences in cytosine modifications between Caucasian and African lymphoblastoid cell lines (LCLs) on the autosomes using whole genome arrays to measure modified cytosines. DNA methylation plays a critical role in establishment and maintenance of X-chromosome inactivation in females. Therefore, we reasoned that by investigating cytosine modification patterns specifically on the X-chromosome, we could obtain valuable information about a chromosome that is often disregarded in genome-wide analyses. We investigated population differences in cytosine modification patterns along the X-chromosome between Caucasian and African LCLs and identified novel sites that escape methylation on the inactive X-chromosome (Xi) in females. We characterized the chromatin state of these loci by incorporating the extensive histone modification ChIP-seq data generated by ENCODE. To explore the relationship between DNA and histone modifications further, we hypothesized that BRD4, a protein that binds acetylated histones, could be preventing some sites from becoming <i>de novo</i> methylated. To test this, we treated 4 female LCLs with JQ1, a small molecule inhibitor of BRD4, but found that JQ1 treatment induced minor changes in cytosine modification levels, and the majority of sites escaping methylation on the Xi remained unmethylated. This suggests that other epigenetic mechanisms or transcription factors are likely playing a larger role in protecting these sites from <i>de novo</i> methylation on the Xi.</p

miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy