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Cellular membranes are, in general, impermeable to macromolecules (herein referred to as macrodrugs, e.g., recombinant protein, expression plasmids, or mRNA), which is a major barrier for clinical translation of macrodrug-based therapies. Encapsulation of macromolecules in lipid nanoparticles (LNPs) can protect the therapeutic agent during transport through the body and facilitate the intracellular delivery via a fusion-based pathway. Furthermore, designing LNPs responsive to stimuli can make their delivery more localized, thus limiting the side effects. However, the principles and criteria for designing such nanoparticles remain unclear. We show that the thermodynamic state of the lipid membrane of the nanoparticle is a key design principle for acoustically responsive fusogenic nanoparticles. We have optimized a cationic LNP (designated LNPLH) with two different phase transitions near physiological conditions for delivering mRNA. A bicistronic mRNA encoding a single domain intracellular antibody fragment and green fluorescent protein (GFP) was introduced into a range of human cancer cell types using LNPLH, and the protein expression was measured via fluorescence corresponding to the GFP expression. The LNPLH/mRNA complex demonstrated low toxicity and high delivery, which was significantly enhanced when the transfection occurred in the presence of acoustic shock waves. The results suggest that the thermodynamic state of LNPs provides an important criterion for stimulus responsive fusogenic nanoparticles to deliver macrodrugs to the inside of cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1driven tumors mostly showed poor ABT-199 responses. Because BCL-2shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.

Mixed Lineage Leukemia(MLL1) translocations encode fusion proteins retaining the N terminus of MLL1, which interacts with the tumor suppressor, menin. This interaction is essential for leukemogenesis and thus is a promising drug target. However, wild-type MLL1 plays a critical role in sustaining hematopoietic stem cells (HSCs); therefore, disruption of an essential MLL1 cofactor would be expected to obliterate normal hematopoiesis. Here we show that rather than working together as a complex, menin and MLL1 regulate distinct pathways during normal hematopoiesis, particularly in HSCs and B cells. We demonstrate the lack of genetic interaction between menin and MLL1 in steady-state or regenerative hematopoiesis and in B-cell differentiation despite the fact that MLL1 is critical for these processes. In B cells, menin- or MLL1-regulated genes can be classified into 3 categories: (1) a relatively small group of coregulated genes including previously described targets Hoxa9and Meis1but also Mecomand Eya1, and much larger groups of (2) exclusively menin-regulated and (3) exclusively MLL1-regulated genes. Our results highlight the large degree of independence of these 2 proteins and demonstrate that menin is not a requisite cofactor for MLL1 during normal hematopoiesis. Furthermore, our data support the development of menin-MLL1–disrupting drugs as safe and selective leukemia targeting agents.

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), including early T-cell precursor ALL (ETP-ALL) cases with poor prognosis. Lmo2 must be recruited to DNA by binding to the hematopoietic basic helix-loop-helix factors Scl/Tal1 or Lyl1. However, it is unknown which of these factors can mediate the leukemic activity of Lmo2. To address this, we have generated Lmo2-transgenic mice lacking either Scl or Lyl1 in the thymus. We show that although Scl is dispensable for Lmo2-driven leukemia, Lyl1 is critical for all oncogenic functions of Lmo2, including upregulation of a stem cell–like gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia. Lyl1 expression is restricted to preleukemic and leukemic stem cell populations in this model, providing a molecular explanation for the stage-specific expression of the Lmo2-induced gene expression program. Moreover, LMO2 and LYL1 are coexpressed in ETP-ALL patient samples, and LYL1 is required for growth of ETP-ALL cell lines. Thus, the LMO2-LYL1 interaction is a promising therapeutic target for inhibiting self-renewing cancer stem cells in T-ALL, including poor-prognosis ETP-ALL cases.

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL), including early T-cell precursor ALL (ETP-ALL) cases with poor prognosis. Lmo2 must be recruited to DNA by binding to the hematopoietic basic helix-loop-helix factors Scl/Tal1 or Lyl1. However, it is unknown which of these factors can mediate the leukemic activity of Lmo2. To address this, we have generated Lmo2-transgenic mice lacking either Scl or Lyl1 in the thymus. We show that although Scl is dispensable for Lmo2-driven leukemia, Lyl1 is critical for all oncogenic functions of Lmo2, including upregulation of a stem cell–like gene signature, aberrant self-renewal of thymocytes, and subsequent generation of T-cell leukemia. Lyl1 expression is restricted to preleukemic and leukemic stem cell populations in this model, providing a molecular explanation for the stage-specific expression of the Lmo2-induced gene expression program. Moreover, LMO2 and LYL1 are coexpressed in ETP-ALL patient samples, and LYL1 is required for growth of ETP-ALL cell lines. Thus, the LMO2-LYL1 interaction is a promising therapeutic target for inhibiting self-renewing cancer stem cells in T-ALL, including poor-prognosis ETP-ALL cases.

Mixed Lineage Leukemia (MLL1) translocations encode fusion proteins retaining the N terminus of MLL1, which interacts with the tumor suppressor, menin. This interaction is essential for leukemogenesis and thus is a promising drug target. However, wild-type MLL1 plays a critical role in sustaining hematopoietic stem cells (HSCs); therefore, disruption of an essential MLL1 cofactor would be expected to obliterate normal hematopoiesis. Here we show that rather than working together as a complex, menin and MLL1 regulate distinct pathways during normal hematopoiesis, particularly in HSCs and B cells. We demonstrate the lack of genetic interaction between menin and MLL1 in steady-state or regenerative hematopoiesis and in B-cell differentiation despite the fact that MLL1 is critical for these processes. In B cells, menin- or MLL1-regulated genes can be classified into 3 categories: (1) a relatively small group of coregulated genes including previously described targets Hoxa9 and Meis1 but also Mecom and Eya1, and much larger groups of (2) exclusively menin-regulated and (3) exclusively MLL1-regulated genes. Our results highlight the large degree of independence of these 2 proteins and demonstrate that menin is not a requisite cofactor for MLL1 during normal hematopoiesis. Furthermore, our data support the development of menin-MLL1–disrupting drugs as safe and selective leukemia targeting agents.

Genotype-specific cancer therapy promises to engender the era of personalised medicines in which rapid identification of tumour specific gene mutations coupled to rapid methods for efficacious drug identification will be applied. Aberrant signal transduction via protein-protein interactions is generally difficult to target with small molecules. However, macromolecules (macrodrugs) can be developed that interfere with protein-protein interactions by binding with high affinity and specificity to contact surfaces. Inhibitors of mutant RAS and its effector protein interactions affect cancer by attenuating aberrant RAS-dependent signal transduction and would be effective against mutant RAS in dividing cells of overt tumours and in putative cancer stem cells when they move into cell-cycle. Results with an antibody fragment blocking effector binding to RAS, illustrates that this is sufficient to prevent cancer. While macrodrugs have inherent problems of bio-distribution and delivery to target cells in patients, their efficacy suggests that efforts to achieve the goal of clinical use should be pursued.

The steps to leukemia following an in utero fusion ofMLL (HRX, ALL-1)to a partner gene in humans are not known. Introduction of theMll-AF9fusion gene into embryonic stem cells results in leukemia in mice with cell-type specificity similar to humans. In this study we used myeloid colony assays, immunophenotyping, and transplantation to evaluate myelopoiesis inMll-AF9mice. Colony assays demonstrated that both prenatal and postnatalMll-AF9tissues have significantly increased numbers of CD11b+/CD117+/Gr-1+/−myeloid cells, often in compact clusters. The self-renewal capacity of prenatal myeloid progenitors was found to decrease following serial replating of colony-forming cells. In contrast, early postnatal myeloid progenitors increased following replating; however, the enhanced self-renewal of early postnatal myeloid progenitor cells was limited and did not result in long-term cell lines or leukemia in vivo. Unlimited replating, long-term CD11b/Gr-1+myeloid cell lines, and the ability to produce early leukemia in vivo in transplantation experiments, were found only in mice with overt leukemia. PrenatalMll-AF9tissues had reduced total (mature and progenitor) CD11b/Gr-1+cells compared with wild-type tissues. Colony replating, immunophenotyping, and cytochemistry suggest that any perturbation of cellular differentiation from the prenatal stage onward is partial and largely reversible. We describe a novel informative in vitroandin vivo model system that permits study of the stages in the pathogenesis ofMllfusion gene leukemia, beginning in prenatal myeloid cells, progressing to a second stage in the postnatal period and, finally, resulting in overt leukemia in adult animals.

The steps to leukemia following an in utero fusion ofMLL (HRX, ALL-1) to a partner gene in humans are not known. Introduction of the Mll-AF9 fusion gene into embryonic stem cells results in leukemia in mice with cell-type specificity similar to humans. In this study we used myeloid colony assays, immunophenotyping, and transplantation to evaluate myelopoiesis in Mll-AF9 mice. Colony assays demonstrated that both prenatal and postnatal Mll-AF9 tissues have significantly increased numbers of CD11b+/CD117+/Gr-1+/− myeloid cells, often in compact clusters. The self-renewal capacity of prenatal myeloid progenitors was found to decrease following serial replating of colony-forming cells. In contrast, early postnatal myeloid progenitors increased following replating; however, the enhanced self-renewal of early postnatal myeloid progenitor cells was limited and did not result in long-term cell lines or leukemia in vivo. Unlimited replating, long-term CD11b/Gr-1+ myeloid cell lines, and the ability to produce early leukemia in vivo in transplantation experiments, were found only in mice with overt leukemia. Prenatal Mll-AF9tissues had reduced total (mature and progenitor) CD11b/Gr-1+ cells compared with wild-type tissues. Colony replating, immunophenotyping, and cytochemistry suggest that any perturbation of cellular differentiation from the prenatal stage onward is partial and largely reversible. We describe a novel informative in vitro and in vivo model system that permits study of the stages in the pathogenesis of Mll fusion gene leukemia, beginning in prenatal myeloid cells, progressing to a second stage in the postnatal period and, finally, resulting in overt leukemia in adult animals.

ABSTRACTChromosomal translocation t(9;11)(p22;q23) in acute myeloid leukemia fuses the MLLand AF9genes. We have inactivated the murine homologue of AF9to elucidate its normal role. No effect on hematopoiesis was observed in mice with a null mutation of Af9. However, an Af9null mutation caused perinatal lethality, and homozygous mice exhibited anomalies of the axial skeleton. Both the cervical and thoracic regions were affected by anterior homeotic transformation. Strikingly, mice lacking functional Af9exhibited a grossly deformed atlas and an extra cervical vertebra. To determine the molecular mediators of this phenotype, analysis of Hoxgene expression by in situ hybridization showed that Af9null embryos have posterior changes in Hoxd4gene expression. We conclude that the Af9gene is required for normal embryogenesis in mice by controlling pattern formation, apparently via control of Hoxgene regulation. This is analogous to the role of Mll,the murine homolog of human MLL, to which the Af9gene fuses in acute myeloid leukemias.

Chromosomal translocation t(9;11)(p22;q23) in acute myeloid leukemia fuses the MLLand AF9genes. We have inactivated the murine homologue of AF9to elucidate its normal role. No effect on hematopoiesis was observed in mice with a null mutation of Af9. However, an Af9null mutation caused perinatal lethality, and homozygous mice exhibited anomalies of the axial skeleton. Both the cervical and thoracic regions were affected by anterior homeotic transformation. Strikingly, mice lacking functional Af9exhibited a grossly deformed atlas and an extra cervical vertebra. To determine the molecular mediators of this phenotype, analysis of Hoxgene expression by in situ hybridization showed that Af9null embryos have posterior changes in Hoxd4gene expression. We conclude that the Af9gene is required for normal embryogenesis in mice by controlling pattern formation, apparently via control of Hoxgene regulation. This is analogous to the role of Mll,the murine homolog of human MLL, to which the Af9gene fuses in acute myeloid leukemias.

Transcription factors are commonly involved in leukemia by activation through chromosomal translocations and normally function in cell type(s) that differ from that of the tumor. TAL2is a member of a basic helix–loop–helix gene family specifically involved in T cell leukemogenesis. Null mutations of Tal2have been made in mice to determine its function during development. Tal2null mutant mice show no obvious defects of hematopoiesis. During embryogenesis, Tal2expression is restricted to the developing midbrain, dorsal diencephalon, and rostroventral diencephalic/telencephalic boundary, partly along presumptive developing fiber tracts. The null mutant mice are viable at birth but growth become progressively retarded and they do not survive to reproductive age. Tal2-deficient mice show a distinct dysgenesis of the midbrain tectum. Due to loss of superficial gray and optical layers, the superior colliculus is reduced in size and the inferior colliculus is abnormally rounded and protruding. Death is most likely due to progressive hydrocephalus which appears to be caused by obstruction of the foramen of Monro (the connection between the ventricles of the forebrain). Thus, in addition to its oncogenicity when ectopically expressed, Tal2normally plays a pivotal role in brain development and without this gene, mice cannot survive to maturity.

Abstract.: LIM-only (LMO) proteins are transcription regulators that function by mediating protein–protein interaction and include the T cell oncogenes encoding LMO1 and LMO2. The oncogenic functions of LMO1 and LMO2 are thought to be mediated by interaction with LDB1 since they form a multimeric protein complex(es). A new member of the Lmo family, Lmo4, has also recently been identified via its interaction with Ldb1. Sequence analysis of the mouse Lmo4 gene shows that it spans about 18 kb and consists of at least six exons, including two alternatively spliced 5′ exons. Unlike Lmo1, the two 5′ exons of Lmo4 do not encode protein. Comparison of the Lmo4 gene structure with the other LMO family members shows the exon structure of Lmo4 differs in the position of exon junctions encoding the second LIM domain and in a novel exon-intron junction at the penultimate codon of the gene. Lmo4 is thus the least conserved known member of the LIM-only family in both nucleotide sequence and exon structure. Physical mapping of the Lmo4/LMO4 genes has shown mouse Lmo4 is located on Chromosome (Chr) 3 and human LMO4 on Chr 1p22.3. This chromosome location is of interest as it occurs in a region that is deleted in a number of human cancers, indicating a possible role of LMO4 in tumorigenesis, like its relatives LMO1 and LMO2.

T cells express either of the two forms of antigen‐specific receptors, the α/β and γ/δ heterodimers. Their structure closely resembles that of immunoglobulins, and the variable part of the receptor molecule is created by somatic assembly of variable, diversity, and joining regions. The genetic structure of T cell receptor (TCR) genes and their rearrangement in T cell development have been elucidated in great detail in recent years. The human genes for the γ and β subunits are located on the short and long arms of chromosome 7, respectively, whereas the δ‐ and α‐chain genes are located in tandem on the centromeric half of the long arm of chromosome 14. Expression of either α/β or 7/6 TCR complexes on T cells in the developing thymus is likely to proceed in an ordered fashion and results in the appearance of distinct T cell subpopulations. The process of DNA rearrangements required for the generation of functional variable region genes also predisposes lymphoid cells to aberrant DNA rearrangements, which can be detected as chromosomal abnormalities such as translocations and inversions. Molecular analysis of such aberrant rearrangements has shown that rearranging loci are fused to loci unrelated to antigen receptor genes. Furthermore, the breakpoint structures represent nonproductive intermediates in the hierachy of physiological rearrangements. Accordingly, T cell tumors arising early in T cell development often carry chromosomal abnormalities involving the δ‐chain locus, whereas tumors generated later in T cell development tend to show aberrations in the α‐chain gene. This pattern seems to reflect the stage‐specific accessibility of TCR loci for rearrangement by the recombinase machinery. This enzyme is guided by specific recombination signals that can sometimes also be found at the site of breakage on the participating locus in chromosomal abnormalities. Although some features of the mechanism of aberrant rearrangements are known, their biological consequences are less well understood. However, molecular analysis of the mechanism of chromosomal aberrations in T cell tumors suggests that their biological consequences may vary. Firm evidence for the pathogenic significance is missing for most of these lesions. This provides a challenge to molecular immunology to determine how chromosomal abnormalities are involved in tumor pathogenesis.— Boehm, T.; Rabbitts, T. H. The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors. FASEB J.3: 2344‐2359; 1989.

FUS(TLS) was first identified as the 5′-part of a fusion gene withCHOP(GADD153, DDIT3) in myxoid liposarcomas with t(12;16)(q13;p11). Homologies were found with theEWSoncogene, which is rearranged in Ewing sarcomas and other neoplasias. The genomic structure ofFUSshows extensive similarities with that ofEWS,but the exon/intron structures differ in the 5′ parts, and overallFUSis smaller thanEWS.Exon 3 ofFUScorresponds to exons 3 and 4 inEWS. FUSexons 4–6 correspond toEWSexons 5–8. Exons 7 to 15 ofFUSare very similar to those inEWS,although theEWSexons are larger than the correspondingFUSexons.FUSandEWSwere expressed in all tissues investigated. The transcripts were stable within the 160-min half-life experiments. No or little variation inFUSorEWSexpression was seen when resting lymphocytes were activated. These observations indicate thatFUSandEWSbelong to the housekeeping type of genes. This view is supported by the presence of the housekeeping gene type of promoter region in both genes.

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VHlocus which accounts for almost all VHsegments known to rearrange in B-lymphocytes. The human germline VHrepertoire consists of approximately 50 functional VHsegments — the exact number depending on the haplotype — spanning 1,100 kilobases upstream of the JHsegments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VHlocus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VHsegments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

ABSTRACTThe HOX11homeobox gene was first identified through studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia. In addition, analysis of Hox11−/−mice has demonstrated a critical role for this gene in murine spleen development. A possible mode of in vivo function for the HOX11 protein in these two situations is regulation of target genes following DNA binding via the homeodomain, but little is known about how HOX11 regulates transcription in vivo. By performing transcriptional studies in yeast and mammalian one-hybrid systems, a modular transcriptional transactivation region at the NH2terminus of HOX11 has been functionally dissected from other parts of the protein. This NH2-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in one-hybrid assays. The importance of the NH2-terminal region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells. Activation of this gene was found to be dependent upon an intact homeodomain in HOX11, but maximal activation was obtained only when the NH2-terminal 50 amino acids of HOX11 was present, showing that this region of HOX11 is important for in vivo transcriptional control of a chromosomal target gene.

ABSTRACTHox11is a homeobox gene essential for spleen formation in mice, since atrophy of the anlage of a developing spleen occurs in early embryonic development in Hox11null mice. HOX11 is also expressed in a subset of T-cell acute leukemias after specific chromosomal translocations. Since the protein has a homeodomain and can activate transcription, it probably exerts at least some of its effects in vivo by regulation of target genes. Representational difference analysis has been used to isolate cDNA clones corresponding to mRNA species activated following stable expression of HOX11 in NIH 3T3 cells. The gene encoding the retinoic acid-synthesizing enzyme aldehyde dehydrogenase 1 (Aldh1), initially called Hdg-1, was found to be ectopically activated by HOX11 in this system. Study ofAldh1gene expression during spleen development showed that the presence of Aldh1mRNA inversely correlated withHox11. Hox11null mouse embryos have elevatedAldh1mRNA in spleen primordia prior to atrophy, whileAldh1seems to be repressed by Hox11during organogenesis of the spleens of wild-type mice. This result suggests that expression of Aldh1 protein is negatively regulated by Hox11 and that abnormal expression of Aldh1 in Hox11null mice may cause loss of splenic precursor cells by aberrant retinoic acid metabolism.

ABSTRACTHox11is a homeobox gene essential for spleen formation in mice, since atrophy of the anlage of a developing spleen occurs in early embryonic development in Hox11null mice. HOX11 is also expressed in a subset of T-cell acute leukemias after specific chromosomal translocations. Since the protein has a homeodomain and can activate transcription, it probably exerts at least some of its effects in vivo by regulation of target genes. Representational difference analysis has been used to isolate cDNA clones corresponding to mRNA species activated following stable expression of HOX11 in NIH 3T3 cells. The gene encoding the retinoic acid-synthesizing enzyme aldehyde dehydrogenase 1 (Aldh1), initially called Hdg-1, was found to be ectopically activated by HOX11 in this system. Study ofAldh1gene expression during spleen development showed that the presence of Aldh1mRNA inversely correlated withHox11. Hox11null mouse embryos have elevatedAldh1mRNA in spleen primordia prior to atrophy, whileAldh1seems to be repressed by Hox11during organogenesis of the spleens of wild-type mice. This result suggests that expression of Aldh1 protein is negatively regulated by Hox11 and that abnormal expression of Aldh1 in Hox11null mice may cause loss of splenic precursor cells by aberrant retinoic acid metabolism.

An inversion of chromosome 14, inv(14)(q11,q32), is frequently observed in human T cell tumors; the cytogenetic breakpoints are of interest because the T cell receptor α-chain and immunoglobulin heavy chain genes reside on chromosome bands 14q11 and 14q32, respectively. We have investigated the structure of the α-chain genes in a T cell line harboring the chromosome 14 inversion. On the normal chromosome 14, a Vαsegment has rearranged nonproductively with a Jαsegment. In contrast, the inverted chromosome features an unprecedented rearrangement in which an Immunoglobulin heavy chain variable gene segment (VH) on chromosome band 14q32 has joined with a Jαsegment from band 14q11. The VH-JαCαrearrangement is productive at the genomic level and therefore may encode a hybrid immunoglobulin/T cell receptor polypeptide.

ABSTRACTThe HOX11homeobox gene was first identified through studies of the t(7;10) and t(10;14) chromosomal translocations of acute T-cell leukemia. In addition, analysis of Hox11-/-mice has demonstrated a critical role for this gene in murine spleen development. A possible mode of in vivo function for the HOX11 protein in these two situations is regulation of target genes following DNA binding via the homeodomain, but little is known about how HOX11 regulates transcription in vivo. By performing transcriptional studies in yeast and mammalian one-hybrid systems, a modular transcriptional transactivation region at the NH2terminus of HOX11 has been functionally dissected from other parts of the protein. This NH2-terminal region includes the previously identified short conserved Hep motif, which itself activates transcription in one-hybrid assays. The importance of the NH2-terminal region for the function of HOX11 in vivo was assayed by activating a HOX11-dependent gene in NIH 3T3 cells. Activation of this gene was found to be dependent upon an intact homeodomain in HOX11, but maximal activation was obtained only when the NH2-terminal 50 amino acids of HOX11 was present, showing that this region of HOX11 is important for in vivo transcriptional control of a chromosomal target gene.

In Burkitt lymphoma cells reciprocal chromosomal translocations are observed between the long arm of chromosome 8 (8q24) and either the long arm of chromosome 14 (14q32), the short arm of chromosome 2 (2p12) or the long arm of chromo some 22 (22q11)1,2. Gene mapping studies have shown that c-myc, the cellular homologue of the viral myc oncogene, is localized at 8q24 (ref 3–5) and that the three immunoglobulin gene loci map at the breakpoints involved in the translocation: immunoglobulin heavy-chain genes are located at 14q32 (refs 6, 7), κ light chains at 2p12 (ref. 8) and λ light chains at, or close to, 22q11 (refs 9,10). This correlation suggests an association of immunoglobulin gene rearrangements with the occurrence of these specific translocations. By using in situ hybridization we have examined the translocation point with respect to c-myc in two cell tines containing 2;8 translocation, and report here that the c-myc gene remains on the chromosome 8 involved in the reciprocal exchange with chromosome 2. We have also confirmed that the c-myc gene moves from the translocated chromosome 8 in a cell line having a 8;14 translocation. These results show that chromosomal breakage can occur on either side of the c-myc gene in Burkitt lymphoma cells.

Protein‐protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS‐effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)‐based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. © 2019 by John Wiley & Sons, Inc.

Recurrent reciprocal chromosomal translocations are present in more than 50% of leukemias. A deeper understanding of how they affect cancer initiation is essential for evaluating the origins of cancer and the potential for therapy based on the translocation products. Mouse models of chromosomal translocations are required for this. Here we summarize three methodologies developed in our laboratory to model chromosomal translocations (knock-in, translocator, and invertor methods). We have used these models to study leukemias caused by fusions of the mixed lineage leukemia (MLL) gene and the Ews-ERG fusion gene to evaluate oncogenicity and elucidate some general principles about translocation products. We show that MLL fusions have the capacity to cause hematopoietic tumors only if expressed in permissive cells and that the Mll-Enl fusion can cause lineage reassignment if the chromosomal translocation occurs in lineage noncommitted progenitors. The leukemia-initiating cells generated by Mll fusions or by Ews-ERG fusion can be committed cells within the hematopoietic pathway. Our translocation mimic models are applicable to any human reciprocal chromosomal translocation.