Correction: SETD2 loss sensitizes cells to PI3Kß and AKT inhibition.

[This corrects the article DOI: 10.18632/oncotarget.26567.].

SETD2 loss sensitizes cells to PI3Kß and AKT inhibition.

Upregulation of the PI3K pathway has been implicated in the initiation and progression of several types of cancer, including renal cell carcinoma (RCC). Although several targeted therapies have been developed for RCC, durable and complete responses are exceptional. Thus, advanced RCC remains a lethal disease, underscoring the need of robust biomarker-based strategies to treat RCC. We report a synthetic lethal interaction between inhibition of phosphatidylinositol 3-kinase beta (PI3Kß) and loss of SETD2 methyltransferase. Clear cell RCC (ccRCC)-derived SETD2 knockout 786-0 and SETD2 mutant A498 cells treated with TGX221 (PI3Kß-specific) and AZD8186 (PI3Kß- and δ-specific) inhibitors displayed decreased cell viability, cell growth, and migration compared to SETD2 proficient 786-0 cells. Inhibition of the p110 δ and α isoforms alone had modest (δ) and no (α) effect on ccRCC cell viability, growth, and migration. In vivo, treatment of SETD2 mutant A498 cells, but not SETD2 proficient 786-0 cells, with AZD8186 significantly decreased tumor growth. Interestingly, inhibition of the downstream effector AKT (MK2206) recapitulated the effects observed in AZD8186-treated SETD2 deficient cells. Our data show that specific inhibition of PI3Kß causes synthetic lethality with SETD2 loss and suggest targeting of the AKT downstream effector pathway offers a rationale for further translational and clinical investigation of PI3Kß-specific inhibitors in ccRCC.

SETD2 Haploinsufficiency for Microtubule Methylation Is an Early Driver of Genomic Instability in Renal Cell Carcinoma.

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.

Methylated α-tubulin antibodies recognize a new microtubule modification on mitotic microtubules.

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.

Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body.

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.

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.

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.

Targeted reduction of KLF6-SV1 restores chemotherapy sensitivity in resistant lung adenocarcinoma.

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.

Molecular cloning of multiple forms of the ovine B7-2 (CD86) costimulatory molecule.

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.

Molecular cloning and mRNA tissue-expression of two isoforms of the ovine costimulatory molecule CD80 (B7-1).

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.