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Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability.Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135–46. ©2018 AACR.

SETD2 loss promotes chromosomal abnormalities.

CENP/B and CREST status of micronuclei

Reduced SETD2 levels promote genomic instability without compromising H3K36 trimethylation.

Multiple Setd2w/f MEF clones display reduced aTubK40me3 protein (A) and relative Setd2 mRNA (B) levels

Human tumor analysis

Micronuclei are a Common Feature among ccRCC-derived Cell Lines.

fig legends for sup figures

Supplementary Figure 7 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 1 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 2 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 6 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure 4 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

Supplementary Figure Legends 1-7 from KLF6-SV1 Is a Novel Antiapoptotic Protein That Targets the BH3-Only Protein NOXA for Degradation and Whose Inhibition Extends Survival in an Ovarian Cancer Model

SummaryMany membrane-less organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The Histone Locus Body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show thatDrosophilaHLBs form through phase separation of the scaffold protein multi-sex combs (Mxc). The size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which catalyzes phase separation, and the nuclear concentration of Mxc, which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Our experiments thus identify a mechanism linking nuclear body growth and size with gene expression.

Loss of the short arm of chromosome 3 (3p) occurs early in >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiquitous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor suppressors as early drivers of tumorigenesis. We previously reported methyltransferase SETD2, which trimethylates H3 histones on lysine 36 (H3K36me3) and is located in the 3p deletion, to also trimethylate microtubules on lysine 40 (αTubK40me3) during mitosis, with αTubK40me3 required for genomic stability. We now show that monoallelic, Setd2-deficient cells retaining H3K36me3, but not αTubK40me3, exhibit a dramatic increase in mitotic defects and micronuclei count, with increased viability compared with biallelic loss. In SETD2-inactivated human kidney cells, rescue with a pathogenic SETD2 mutant deficient for microtubule (αTubK40me3), but not histone (H3K36me3) methylation, replicated this phenotype. Genomic instability (micronuclei) was also a hallmark of patient-derived cells from ccRCC. These data show that the SETD2 tumor suppressor displays a haploinsufficiency phenotype disproportionately impacting microtubule methylation and serves as an early driver of genomic instability. Significance: Loss of a single allele of a chromatin modifier plays a role in promoting oncogenesis, underscoring the growing relevance of tumor suppressor haploinsufficiency in tumorigenesis. Cancer Res; 78(12); 3135–46. ©2018 AACR.

Concentrating factors in nuclear bodies is thought to promote efficient gene expression. Tatomer et al. show that the histone locus body (HLB) concentrates pre-mRNA processing factors at replication-dependent histone genes, resulting in optimal 3′ end formation of histone mRNAs coupled with transcription termination., The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination.

Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of α-tubulin, which is trimethylated (α-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (α-TubK40me3 pAb) and monoclonal (α-TubK40me3 mAb) antibodies to a methylated α-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated α-tubulin migrated faster than unmethylated α-tubulin, suggesting methylation may be a signal for additional processing of α-tubulin and/or microtubules. As the first reagents that specifically recognize methylated α-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.

Mammalian cell identity or cell fate is governed by transcription factors that recruit co-activators to regulate cell-specific gene expression programs. The histone acetyltransferase (HAT) paralogs EP300 and CREB binding protein (CBP) are master transcriptional co-activators that catalyze acetylation of key histones that mark active enhancers, thereby operating at a fundamental step of transcriptional control. Enhancers are often co-opted by cancer cells to control expression of oncogenic transcriptional programs. Identifying cancer-specific dependencies will greatly help prioritize clinical indications that are uniquely vulnerable to HAT inhibition, allowing for precise targeting of cancer cells while sparing normal tissue to maximize the therapeutic window and clinical efficacy. Estrogen receptor (ER) signaling is a validated clinical pathway in over 70% of breast cancer. ER-directed therapies are foundational for the treatment of ER+ breast cancer. However, these therapies are limited by residual ER signaling, which remains online through late-stage disease in a significant fraction of patients. EP300/CBP are critical components of the core ER signaling complex, which is recruited to target DNA by ER and other coactivators, to potentiate ER signaling. Furthermore, CBP and EP300 are dependency factors in the context of ESR1 mutations that arise in metastatic disease to facilitate disease progression. We report for the first time the activity of a novel, potent, selective and in vivo active EP300/CBP HAT inhibitor for the treatment of ER-positive (ER+) breast cancer. CPI-1 has pM biochemical and nM activity in ER+ breast cancer cell lines. Analysis of nascent and steady-state RNA levels facilitated by PRO-seq and RNA-seq technologies revealed a time and dose-dependent suppression of the ER transcriptome. Dynamic gene regulation was coupled with enhancer RNA (eRNA) transcription and enhancer acetylation, to identify enhancers that were hypersensitive to CBP/EP300 HAT inhibition. CPI-1 demonstrates significant tumor growth inhibition in a xenograft mouse model of ER+ breast cancer as a monotherapy and together with an ER targeting agent, the combination displays superior efficacy to either treatment alone. These results highlight the potential of targeting key oncogenic transcriptional nodes with complementary therapeutic mechanisms to achieve superior results and reveals the potential utility of inhibiting the HAT domain of EP300/CBP in molecularly refined cancer populations. Citation Format: Archana Bommi-Reddy, Jonathan Wilson, Annissa J. Huhn, Esteban Terzo, Florence Poy, Sungmi Park, Michael J. Steinbaugh, Barbara M. Bryant, Andrew R. Conery, Richard Cummings, Julian Levell, Robert J. Sims. Efficacy of a novel EP300/CBP histone acetyltransferase inhibitor in hormone responsive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4722.

Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21–22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.

Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer for which several new targeted therapies exist. However, responses of primary clear cell RCC (ccRCC) tumors and metastases to these targeted agents are variable, and complete responses are relatively uncommon underscoring the need to identify reliable biomarkers to predict tumor behavior. Bi-allelic inactivation of the tumor suppressor gene SETD2, encoding a lysine methyltransferase, occur in approximately 20% of ccRCC tumors while the SETD2 protein deficiency rate has been reported to be as high as 34%. The phosphatidylinositide 3-kinases (PI3Ks) have emerged as important therapeutic targets for the treatment of solid and hematological malignancies, including ccRCC. Here, we show that genetic and pharmacologic inhibition of the PI3Kβ isoform results in inhibition of SETD2 deficient (-/-) ccRCC-derived 786-O and A498 cells, which exhibit increased sensitivity to PI3Kβ-specific inhibitors TGX221 (IC50 = 2.3 - 4.5 µM, respectively) and AZD8186 (IC50 = 1.79 - 0.24 µM, respectively) as compared to SETD2 proficient (+/+) 786-O cells (IC50 = >10 µM). Inhibition of the p110δ isoform alone had a modest effect on ccRCC cell growth and migration. Importantly, genetic (with siRNA) and pharmacologic (with BYL719) inhibition of p110α had no effect on the ccRCC cell lines. Inhibition of AKT with the small molecule inhibitor MK2206 recapitulated the effects seen with AZD8186 in ccRCC cell lines with SETD2 loss, suggesting PI3Kβ mediates the synthetic lethality via AKT signaling in the context of SETD2 loss in ccRCC. Our work strongly suggests a synthetic lethal-type of interaction between PI3Kβ inhibition and SETD2 loss and offers a rationale for further translational and clinical investigation of small molecule inhibitors of PI3Kβ in RCC. Xenograft studies are ongoing to investigate the in vivo efficacy of AZD8186. Citation Format: Esteban Terzo, Yun C. Chiang, Anna Chytil, Valerie M. Jansen, Kimryn Rathmell. Synthetic lethal interaction between SETD2 loss and inhibition of PI3Kβ in clear cell renal cell carcinoma (ccRCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1942.

Introduction: Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer for which several new targeted therapies exist. However, responses of primary clear cell RCC (ccRCC) tumors and metastases to these targeted agents are variable, and complete responses are relatively uncommon, underscoring the need to identify reliable biomarkers to predict tumor behavior. Mutations in the tumor suppressor gene SETD2, encoding a lysine methyltransferase, occur in 3-16% of ccRCC tumors while the SETD2 protein deficiency rate has been reported to be as high as 34%. The phosphatidylinositide 3-kinases (PI3Ks) have emerged as important therapeutic targets for the treatment of solid and hematologic malignancies, including ccRCC. In this study, we determine the extent to which small-molecule inhibitors against the p110α, p110β, and p110δ isoforms of PI3K inhibit growth of RCC with SETD2 loss. Methods: RNAi oligonucleotides and small-molecule inhibitors targeting the p110α (BYL719), p110β (TGX221, AZD8186), and p110δ (idelalisib) isoforms of PI3K were tested against ccRCC cell lines with SETD2 loss. The effect of drugs on cell growth and survival was determined by (1) proliferation in monolayer, (2) 3D growth in Matrigel, and (3) cell migration in wound healing assay. Downstream signaling pathways were evaluated by immunoblot analysis. Results: Genetic and pharmacologic inhibition of p110β resulted in inhibition of ccRCC cell lines with SETD2 loss. ccRCC-derived SETD2 deficient (-/-) 786-O and A498 cells exhibited increased sensitivity to TGX221 (IC50 = 2.3 - 4.5 µM, respectively) and AZD8186 (IC50 = 1.79 - 0.24 µM, respectively) as compared to SETD2 proficient (+/+) 786-O cells (IC50 = >10 µM). Inhibition of the p110δ isoform alone had a modest effect on ccRCC cell growth and migration. Importantly, genetic (with siRNA) and pharmacologic (with BYL719) inhibition of p110α had no effect on the ccRCC cell lines. Inhibition of AKT with the small-molecule inhibitor MK2206 recapitulated the effects seen with AZD8186 in ccRCC cell lines with SETD2 loss, suggesting PI3Kβ mediates the synthetic lethality via AKT signaling in the context of SETD2 loss in ccRCC. Conclusions: Our work strongly suggests a synthetic lethal-type of interaction between PI3Kβ inhibition and SETD2 loss and offers a rationale for further translational and clinical investigation of small-molecule inhibitors of PI3Kβ in RCC. Xenograft studies are ongoing to investigate the in vivo efficacy of AZD8186. Citation Format: Esteban Terzo, Valerie M. Jansen, Yun-Chen Chiang, Kimryn W. Rathmell. SETD2-PI3Kβ synthetic lethality [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B059.

Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Here, we demonstrate that chemoresistant lung cancer cells express increased levels of KLF6-SV1. Furthermore, targeted reduction of KLF6-SV1 using RNA interference restores chemotherapy sensitivity to lung cancer cells both in culture and in vivo through induction of apoptosis. Conversely, overexpression of KLF6-SV1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model. Combined, these findings highlight a functional role for the KLF6-SV1 splice variant in the regulation of chemotherapy response in lung cancer and could provide novel insight into lung cancer therapy.

Defects in apoptosis are not only a hallmark of cancer initiation and progression but can also underlie the development of chemoresistance. How the tightly regulated cascade of protein-protein interactions between members of three competing protein families regulating the apoptotic cascade is subverted in tumor cells is incompletely understood. Here, we show that KLF6-SV1, whose overexpression is associated with poor survival in several different cancers and is an alternatively spliced isoform of the Krüppel-like tumor suppressor KLF6, is a critical prosurvival/antiapoptotic protein. KLF6-SV1 binds the proapoptotic BH3-only protein NOXA, which results in their mutual HDM2-dependent degradation. In turn, this increases the intracellular concentration of the prosurvival binding partner of NOXA, Mcl-1, and effectively blocks apoptosis. In an ovarian cancer model, systemically delivered small interfering RNA against KLF6-SV1 induces spontaneous apoptosis of tumor cells, decreases tumor burden, and restores cisplatin sensitivity in vivo. Moreover, i.p. delivery of siKLF6-SV1 RNA halts ovarian tumor progression and improves median and overall survival (progression-free for >15 months; P < 0.0002) in mice in a dose-dependent manner. Thus, KLF6-SV1 represents a novel regulator of protein interactions in the apoptotic cascade and a therapeutically targetable control point. [Cancer Res 2009;69(11):4733–41]

The Drosophila Multi Sex Combs (Mxc) protein is necessary for the recruitment of histone mRNA biosynthetic factors to the histone locus body (HLB). Mxc contains multiple domains required for HLB assembly and histone mRNA biosynthesis. Two N-terminal domains of Mxc are essential for promoting HLB assembly via a self-interaction., Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.

Epigenetic regulators are emerging as major drivers of renal cell carcinoma (RCC). SETD2, a tumor suppressor commonly mutated in RCC, is the sole enzyme responsible for tri-methylating histone H3 on lysine 36 (H3K36me3) of actively transcribed genes. We recently discovered that SETD2 also trimethylates the non-histone target α-tubulin on lysine 40 (α-TubK40Me3) of mitotic microtubules. We have demonstrated that mutations altering SETD2’s ability to trimethylate H3K36 and α-TubK40 are associated with genomic instability in cancer progression. We hypothesized that defective SETD2 methylating activity gives rise to deleterious cell signaling cascades that promote the tumorigenic features of SETD2 loss. Preliminary data show that RCC-derived 786-0 cells that are deleted for SETD2 (SETD2-KO) or harbor pathogenic mutations are more sensitive to treatment with the PI3Kβ-specific inhibitor TGX-221 than their wild type counterparts. Phosphatidylinositol 3kinases (PI3Ks) are a family of lipid kinases that coordinate signals from growth factors, cytokines and other environmental cues, translating them into intracellular signals controlling diverse signaling pathways. Several members of the PI3K/AKT signaling pathway, including the PI3K α and β isoforms, are frequently mutated in a variety of cancers, making this pathway a prime drug target for anti-cancer therapy. These pathways control numerous biological processes, such as cell proliferation, growth, and motility. Examination of structurally variant SETD2 mutants suggests that the loss of SETD2 methylating activity mediates this interaction, and future studies will examine the functional consequences of inhibiting this pathway. In summary, these data suggest a synthetic-lethal type of interaction between SETD2 loss (and specifically loss of methylating activity) and an aberrant PI3K/AKT pathway. Citation Format: Esteban Terzo, Jeanne Chiang, W. Kimryn Rathmell. Exploring the molecular mechanism underlying SETD2-PI3Kβ synthetic-lethal interaction in renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2017-3071

To facilitate analysis of the role of costimulatory molecules in the ovine immune system, we cloned and sequenced eight putative alternatively spliced transcripts of the sheep CD86 (B7-2) costimulatory molecule. Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned the sheep CD86 (B7-2) molecule that encodes eight forms, which differ in the length of the signal peptide, the presence or absence of a transmembrane region and in their cytoplasmic tails. Comparison of the deduced amino acid sequence of the largest ovine CD86 TM form (CD86-2) with the sequence of cattle, pig, human and mouse CD86 indicated that the deduced protein had a higher degree of similarity to cattle (85% of amino acid identity) than to pig (77%), human (59%), and mouse sequence (45% of identity). Our results indicate that mRNA transcripts encoding different CD86 protein forms are expressed in sheep, like in other mammals, and suggest that the expression of this gene may be regulated at the transcriptional or RNA splicing level, which could give rise to tissue-specific expression of CD86. It is possible that, in the sheep, these CD86 mRNA variants could play different regulatory roles in T cell activation.

Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned two putative alternatively spliced transcripts of the sheep CD80 (B7-1) molecule that encode both transmembrane (TM) and secreted (s) forms of CD80 protein. Comparison of the amino acid sequence of the TM form of ovine CD80 with the sequence of cattle, swine and human CD80 indicated that the deduced protein had a higher degree of similarity to cattle (87% of amino acid identity) than to pig (68%) and human sequence (53% of homology). In tissues, RT-PCR using primers for the TM and the sCD80 transcripts indicated that the expression of both CD80 transcripts was almost exclusively expressed in the hematolymphoid system, with the exception of the uterus. The sCD80 transcript was expressed in peripheral blood mononuclear cells (PBMC), uterus and lymph node, whereas the TM-CD80 transcript was very weakly detected only in PBMC cells. Our result indicates that mRNA transcripts encoding both membrane-bound and secreted CD80 proteins are expressed in sheep like in other animals. However, in contrast with the CD80 molecules from other species, the secreted form of sheep CD80 seems to be the predominant form expressed in the ovine PBMC and other tissues, suggesting that the TM-CD80 represents a rare transcript in this species. Interestingly, the expression of both forms of the CD80 molecule was not affected by treatment of sheep PBMC with Concanavalin A (ConA), as detected by RT-PCR. This is the first report describing the identification of a B7 costimulatory transcript in sheep.

Hereditary cancer syndromes represent a powerful and tractable biologic system for identifying cancer-causing genes. Though the syndromes themselves may be rare, their study can provide insights into the basis of the more common sporadic forms of the cancer. Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal dominant bone dysplasia / bone cancer syndrome. This hereditary cancer syndrome is characterized by bone infarctions, cortical growth abnormalities, and pathologic fractures. Most notably, 35% of affected individuals develop bone MFH, a sarcoma that in its sporadic form accounts for 6% of all bone cancers and is believed to be etiologically related to osteosarcoma. Indeed, one of our affected family members developed histologically-proven osteosarcoma, thus further supporting a genetic link between these tumor types. Using a linkage based approach, we previously mapped the DMS-MFH tumor suppressor gene locus to chromosome 9p21-22 (1) and then through LOH analysis demonstrated that hereditary and sporadic forms of MFH most likely share a single underlying genetic etiology (2). We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously unrecognized terminal exons of the methylthioadenosine phosphorylase (MTAP) gene. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine/methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Intriguingly, two of the novel MTAP exons arose from early and independent retroviral integration events in primate genomes at least 40 MYR ago and since their genomic integration have gained a functional role. Six distinct retroviral-sequence containing MTAP isoforms, each of which can physically interact with archetype MTAP (i.e., exons 1-8), are identified. The disease-causing / cancer-associated mutations occur within one of these retroviral-derived exons. The mutations result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Based on these findings in a hereditary form of bone sarcoma, we then analyzed the expression of these MTAP isoforms in a sample set (n=16) of sporadic osteosarcoma samples. All tumor samples expressed similar levels of the archetype MTAP RNA sequence but the expression pattern of the splice variants varied markedly between nearly all the samples. The majority of samples did not express SV1 (n=11/16) and nearly half did not express SV6 (n=9/16). Taken together, these results identify the first gene involved in the development of bone MFH / osteosarcoma and have potential implications for the treatment of this human cancer. References: 1. Am J Hum Genet. 64:801-7; 1999. 2. Genes Chromosomes Cancer. 27:191-5; 2000. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-89. doi:1538-7445.AM2012-LB-89