George P. Souroullas

Adult Hematopoietic Stem and Progenitor Cells Require Either Lyl1 or Scl for Survival

Scl and Lyl1 encode two related basic-helix-loop-helix transcription factors implicated in T cell acute lymphoblastic leukemia. Previous studies showed that Scl is essential for embryonic and adult erythropoiesis, while Lyl1 is important for B cell development. Single-knockout mice have not revealed an essential function for Scl or Lyl1 in adult hematopoietic stem cells (HSCs). To determine if maintenance of HSCs in single-knockout mice is due to functional redundancy, we generated Lyl1;Scl-conditional double-knockout mice. Here, we report a striking genetic interaction between the two genes, with a clear dose dependence for the presence of Scl or Lyl1 alleles for HSC function. Bone marrow repopulation assays and analyses demonstrated rapid loss of hematopoietic progenitors due to apoptosis. The function of HSCs could be rescued by a single allele of Lyl1 but not Scl. These results show that expression of at least one of these factors is essential for maintenance of adult HSC function.

An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation.

B cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase (EZH2), but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic Ezh2 gain-of-function mutation (EZH2Y646F in human; Ezh2Y641F in mouse) is conditionally expressed. Expression of Ezh2Y641F in mouse B cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Overexpression of the anti-apoptotic protein Bcl2, but not the oncoprotein Myc, or loss of the tumor suppressor protein p53 (encoded by Trp53 in mice) further accelerated lymphoma progression. Expression of the mutant Braf but not the mutant Nras oncoprotein further accelerated melanoma progression. Although expression of Ezh2Y641F globally increased the abundance of trimethylated Lys27 of histone H3 (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 that was associated with increased transcription at many loci. These results suggest that Ezh2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure, inducing both repression and activation of polycomb-regulated loci.B-cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase, but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic EZH2 gain-of-function mutation (Y646F in human, Y641F in the mouse) can be conditionally expressed. Expression of Ezh2Y641F in mouse B-cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Bcl2 overexpression or p53 loss, but not c-Myc overexpression, further accelerated lymphoma progression, and expression of mutant B-Raf but not mutant N-Ras further accelerated melanoma progression. Although expression of Ezh2Y641F increased abundance of global H3K27 trimethylation (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 associated with increased transcription at many loci. These results suggest that Ezh2Y641F induces lymphoma and melanoma through a vast reorganization of chromatin structure inducing both repression and activation of polycomb-regulated loci.

The transcription factor Lyl-1 regulates lymphoid specification and the maintenance of early T lineage progenitors

Thymopoiesis depends on the recruitment and expansion of bone marrow–derived progenitor populations; tight regulation of these processes is required for maintenance of the homeostasis of the T lineage. Lyl-1, a transcription factor that regulates hematopoietic progenitors, is expressed in thymocyte progenitors until T cell commitment. Here we demonstrate a requirement for Lyl-1 in lymphoid specification and the maintenance of early T lineage progenitors (ETPs). Lyl-1 deficiency resulted in profound defects in the generation of lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and ETPs. Lyl-1-deficient ETPs and thymocyte progenitors at the CD4−CD8− double-negative 2 (DN2) stage showed more apoptosis, blocked differentiation and impaired population expansion. We identified Gfi1 as a critical transcriptional target of Lyl-1-mediated lymphopoiesis of T cells. Thus, Lyl-1 is a pivotal component of a transcriptional program that controls the lymphoid specification and maintenance of ETPs.

A new allele of Lyl1 confirms its important role in hematopoietic stem cell function

Lyl1 codes for a bHLH protein that is an important regulator of hematopoietic stem cell function. An existing mutant allele of Lyl1 features a lacZ gene inserted in‐frame into the fourth exon, leaving behind the N‐terminus and the DNA‐binding basic region, resulting in a translated chimeric protein. Here, we have generated a null allele, which allowed us to examine residual function of the N‐terminus in the absence of a bHLH region. The new Lyl1‐/‐ mouse model exhibited a reduced ability to generate lymphoid lineages and a somewhat more severe hematopoietic repopulation defect when transplanting purified hematopoietic stem cells. Our data show that in the absence of the HLH but presence of the N‐terminus, residual function of the Lyl1 is detectable but relatively minor. The new model may be of use for studies of Lyl1 in which a null allele is required, or for which presence of the LacZ may complicate the combined use of additional mouse models bearing the lacZ marker. genesis 49:441–448, 2011.

Stem cells: Down's syndrome link to ageing

Triplication of the enzyme USP16 in models of Downs syndrome creates defects in the stem cells resident in adult tissues. This finding provides insight into stem-cell homeostasis during ageing. See Article p.380 People with Downs syndrome have abnormalities in multiple tissues including mental retardation and early ageing. The disease is often the result of full or partial trisomy of chromosome 21, but the molecular mechanisms underlying the observed cellular defects remain largely unknown. An analysis of haematopoietic stem cells in the Downs syndrome mouse model Ts65Dn has revealed a reduced self-renewal associated with the proliferation of cells expressing three copies of the Usp16 gene, which encodes a deubiquitination enzyme involved in chromatin remodelling and cell cycle progression. In a second Downs syndrome mouse model, Ts1Cje, haematopoietic stem cells were not defective. Downregulation of USP16 rescued the functional defects of affected Ts65Dn cells. Overexpression of USP16 in normal human fibroblasts reduced their proliferative capacity and USP16 downregulation partially rescued human Downs syndrome fibroblast proliferation defects. The authors propose that USP16 is a potential target for therapeutics designed to ameliorate the pathologies associated with this syndrome.

The expansion of T-cells and hematopoietic progenitors as a result of overexpression of the lymphoblastic leukemia gene, Lyl1 can support leukemia formation

This study investigates the function of the lymphoblastic leukemia gene, Lyl1 in the hematopoietic system and its oncogenic potential in the development of leukemia. Overexpression of Lyl1 in mouse bone marrow cells caused T-cell increase in the peripheral blood and expansion of the hematopoietic progenitors in culture and in the bone marrow. These observations were the result of increased proliferation and suppressed apoptosis of the progenitor cells caused by the Lyl1-overexpression. Our studies present substantial evidence supporting the secondary, pro-leukemic effect of Lyl1 in early hematopoietic progenitors with the potential to cause expansion of malignant cells with a stem/early progenitor-like phenotype.

Ataxin1L Is a Regulator of HSC Function Highlighting the Utility of Cross-Tissue Comparisons for Gene Discovery

Hematopoietic stem cells (HSCs) are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene Ataxin-1-like (Atxn1L) was highly expressed in HSCs, we examined its role in HSC function through in vitro and in vivo assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated Atxn1L null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that Atxn1L is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein–protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.

Lkb1 deletion in murine B lymphocytes promotes cell death and cancer

LKB1 (also known as STK11) is a potent tumor suppressor in solid tumors, such as melanoma and lung adenocarcinoma, but inactivation in hematopoietic cells causes cell death without signs of tumorigenesis. We noted somatic LKB1 deletion or mutation at low frequency in human B-cell lymphoma. To determine if LKB1 inactivation is a passenger or driver event in lymphoid cancers, we examined the effects of conditional inactivation of Lkb1 in murine lymphocytes. Consistent with prior reports, Lkb1 deletion in either T or B cells resulted in massive, lineage-specific apoptosis. Surprisingly, despite an 80% reduction of peripheral B-cell number, animals harboring somatic B-lineage Lkb1 deletion developed aggressive B-cell lymphoma with high penetrance and moderate latency. Malignant B cells exhibited somatic Lkb1 recombination. In contrast, Lkb1 deletion in T cells did not promote tumorigenesis. Concomitant Ras activation with Lkb1 deletion reduced T-cell apoptosis, but did not enhance tumor formation in T or B cells. These results suggest that although physiologic LKB1 expression exerts a potent pro-survival effect in lymphocytes, LKB1 inactivation nonetheless facilitates transformation of B, but not T, lymphocytes.

Abstract 1087: Differential oncogenicity of N-RAS mutations in melanoma.

In human cancer, N-RAS mutations resulting in constitutive, oncogenic signaling are predominately localized to codons 12, 13 or 61. Traditionally, activating RAS mutations have been considered oncogenically equivalent, yet recent studies suggest important clinical distinctions between colon cancers containing K-RAS codon 12 vs. codon 13 mutations. More than 20% of human melanomas harbor N-RAS mutations, the vast majority of which target codon 61, as opposed to N-RAS mutations of codon 12 or 13, which are common in other tumor types. To address this issue, we characterized syngeneic knock-in mouse models conditionally expressing either N-Ras G12D or N-Ras Q61R under the control of the endogenous locus. In primary melanocyte cultures, activation of either N-Ras allele reduced cellular proliferation even in the presence of concomitant p16 INK4a deletion. Correspondingly, melanocyte-specific N-Ras G12D expression combined with p16 INK4a loss failed to efficiently promote melanoma formation in mice. In contrast, melanocyte-specific N-Ras Q61R induction readily combined with p16 INK4a loss to promote melanoma formation in vivo with short latency and high penetrance. These results provide a first murine model of melanoma featuring knock-in N-Ras mutation Q61R , and suggest that the preference for codon 61 mutations in human tumors is conserved in mice. Citation Format: Christin Burd, William R. Jeck, Kailing Fu, Kelly S. Clark, Jessie C. Xiong, George P. Souroullas, Norman E. Sharpless. Differential oncogenicity of N-RAS mutations in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2013-1087