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Background: Adoptive cell therapies using Chimeric Antigen Receptors (CARs) show durable clinical benefit for patients with hematologic malignancies, however, challenges remain for enabling this personalized treatment to be delivered in a timely, cost effective, and logistically friendly manner. Methods: Lentiviral vectors (LV) encoding CD19 and CD22 CARs with a synthetic driver element were packaged with a VSV-G envelope designed with the capability to target and activate CD3pos T cells in whole blood. LV were directly reconstituted in peripheral whole blood for four hours and total nucleated cells (TNC) or peripheral blood mononuclear cells (PBMC) were rapidly isolated utilizing two different closed systems. Immediately following the four-hour exposure event, isolated TNC or PBMC were injected subcutaneously into mice with disseminated Raji luciferase tumor cells. For further characterization, LV-exposed TNC and PBMC were cultured in vitro for six days and functionally examined. Results: Following four-hour exposure to CD3-directed LVs, more than 90% of T cells, including naïve/naïve derived (CCR7pos CD45ROneg) and central memory (CCR7pos CD45ROpos) T cells present within isolated TNC or PBMC exhibited a significant decrease in CD3 surface expression. Subcutaneous injection of gene modified TNC or PBMC resulted in the in vivo generation and expansion of large numbers of circulating CAR-T positive cells with complete eradication of disseminated Raji tumors. In parallel cell culture experiments, TNC or PBMC isolated following four-hour LV whole blood exposure exhibited robust expansion without additional T-cell receptor (TCR) or CD3 stimulation, while TNC or PBMC not exposed to virus did not show any expansion. Following six days in culture, immunophenotyping by flow cytometry demonstrated that more than 90% of the cells were CD8pos and CD4pos T cells with CAR-T expression present on central memory (CCR7pos CD45ROpos) and effector memory (CCR7neg CD45ROpos) T cells. CAR-T antigen specificity to CD19 and CD22 was measured by IFN-gamma release co-culture assays. Conclusion: We conclude that large numbers of functionally active CAR-T positive cells can be generated both in vitro and in vivo following a four-hour peripheral whole blood exposure to CD3-directed LVs encoding for CARs with a synthetic driver element. These results provide the basis for an autologous same-day peripheral blood draw to subcutaneous injection rapid point-of-care (rPOC) approach. Citation Format: Dongming Zhang, Frederic Vigant, Qun He, Anirban Kundu, Wei Zhang, Hongliang Zong, Ewa Jaruga-Killeen, Gregory Schreiber, Michelle Andraza, Alissa R. Kerner, Gregory I. Frost, Sid P. Kerkar. Subcutaneous injection of total nucleated cells rapidly isolated following four-hour peripheral whole blood exposure to CD3-directed CAR-T lentiviruses with a synthetic driver results in robust CAR-T proliferation and anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1511.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFβ and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell’s sensitivity to Hsp90 inhibition.

CDK11(p58), a member of the p34(cdc2)-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11(p58) interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11(p58) was autophosphorylated, and the main functions of CDK11(p58), such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11(p58) was autophosphorylated at Thr-370. By mutagenesis, we created CDK11(p58) T370A and CDK11(p58) T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11(p58), respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11(p58) and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivation of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11(p58). Moreover, Thr-370 mutants might affect CDK11(p58)-mediated signaling pathways.

ATF5, a member of activating transcription factor (ATF)/cAMP-response element-binding protein (CREB) family of b-ZIP transcription factors, contributes to neural cell differentiation and is involved in cell apoptosis in response to cisplatin and a number of environment factors. However, the mechanisms governing the regulation of ATF5 protein during apoptosis are largely unknown. In this study we reported that ATF5 protein was a substrate of the ubiquitin-proteasome pathway. Interestingly, the ubiquitin-dependent degradation of exogenous ATF5 protein was independent of lysine residues. Instead, the addition of a large N-terminal enhanced green fluorescence protein tag increased the stability of ATF5 protein, and the free amino acid group of the N-terminal methionine of ATF5 protein was a site for ubiquitinylation, indicating that exogenous ATF5 was degraded via the ubiquitin-proteasome system through N-terminal ubiquitinylation. Furthermore, cisplatin increased ATF5 protein expression via preventing its ubiquitin-dependent degradation, which might be associated with its promoting the nucleus-to-cytoplasm translocation of E2 ubiquitin-conjugating enzyme Cdc34 and reducing the interaction between ATF5 and Cdc34. In summary, a down-regulation of proteasome-mediated degradation of ATF5 might contribute to cisplatin-induced apoptosis, providing a new mechanism of cisplatin-induced apoptosis.

CDK11(p58), a 58kDa protein of the PITSLRE kinase family, plays an important role in cell cycle progression, and is closely related to cell apoptosis. To gain further insight into the function of CDK11(p58), we screened a human fetal liver cDNA library for its interacting proteins using the yeast two-hybrid system. Here we report that histone acetyltransferase (HAT) HBO1, a MYST family protein, interacts with CDK11(p58) in vitro and in vivo. CDK11(p58) and HBO1 colocalize in the cell nucleus. Recombinant CDK11(p58) enhances the HAT activity of HBO1 significantly in vitro. Meanwhile, overexpression of CDK11(p58) in mammalian cells leads to the enhanced HAT activity of HBO1 towards free histones. Thus, we conclude that CDK11(p58) is a new interacting protein and a novel regulator of HBO1. Both of the proteins may be involved in the regulation of eukaryotic transcription.

The elevated levels of β1,4-galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5′-region flanking the transcription start point of the GalT I gene (–1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides –205 and –200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the β1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.

It is known that small glutamine-rich TPR-containing protein (SGT) is the member of TPR motif family. However, the biological functions of SGT remain unclear. In this paper, we report that SGT plays a role in apoptotic signaling. Ectopic expression of SGT enhances DNA fragment and nucleus breakage after the induction of apoptosis. Increasing mRNA level of SGT is also observed in 7721 cells undergoing apoptosis, knockdown the expression of endogenous SGT contributes to the decrease of apoptosis of 7721 cells. Deletion analysis reveals that TPR domain is critical to pro-apoptotic function of SGT. Furthermore, we demonstrated that the PARP cleavage and cytochrome c release are enhanced when SGT is overexpressed in 7721 cells during apoptosis. Collectively, our results indicate that SGT is a new pro-apoptotic factor.

Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase 4 (CDK 4) and CDK 6, but also in many other aspects such as cell cycle, cell differentiation, transcriptional regulation and apoptosis. In this work, we screened a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain. Immunofluorescence experiments showed that eIF3k distributed both in nucleus and cytoplasm and colocalized with cyclin D3. In addition, the cellular translation activity in HeLa cells was upregulated by cyclin D3 overexpression and the mRNA levels are constant. These data provide a new clue to our understanding of the cellular function of cyclin D3.

The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases. During apoptosis induced by some stimuli, specific PITSLRE isoforms are cleaved by caspase to produce a protein that contains the C-terminal kinase domain of the PITSLRE proteins (p110C). The p110C induces apoptosis when it is ectopically expressed in Chinese hamster ovary cells. In our study, similar induction of this p110C was observed during anoikis in NIH3T3 cells. To investigate the molecular mechanism of apoptosis mediated by p110C, we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified p21-activated kinase 1 (PAK1) as an interacting partner of p110C. The association of p110C with PAK1 was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscope analysis. The interaction of p110C with PAK1 occurred within the residues 210-332 of PAK1. Neither association between p58PITSLRE or p110PITSLRE and PAK1 nor association between p110C and PAK2 or PAK3 was observed. Anoikis was increased and PAK1 activity was inhibited when NIH3T3 cells were transfected with p110C. Furthermore, the binding of p110C with PAK1 and inhibition of PAK1 activity were also observed during anoikis. Taken together, these data suggested that PAK1 might participate in the apoptotic pathway mediated by p110C.

SummaryChromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated amongst these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappaB pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.

The androgen-signaling pathway plays critical roles in normal prostate development, benign prostatic hyperplasia, established prostate cancer, and in prostate carcinogenesis. In this study, we report that trihydrophobin 1 (TH1) is a potent negative regulator to attenuate the androgen signal-transduction cascade through promoting androgen receptor (AR) degradation. TH1 interacts with AR both in vitro and in vivo, decreases the stability of AR, and promotes AR ubiquitination in a ligand-independent manner. TH1 also associates with AR at the active androgen-responsive prostate-specific antigen (PSA) promoter in the nucleus of LNCaP cells. Decrease of endogenous AR protein by TH1 interferes with androgen-induced luciferase reporter expression and reduces endogenous PSA expression. Taken together, these results indicate that TH1 is a novel regulator to control the duration and magnitude of androgen signal transduction and might be directly involved in androgen-related developmental, physiological, and pathological processes.

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11p58 as a novel protein involved in the regulation of VDR. CDK11p58, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11p58 interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11p58 decreased the stability of VDR through promoting its ubiquitin–proteasome-mediated degradation. Taken together, these results suggest that CDK11p58 is involved in the negative regulation of VDR.

BCL2L12, a newly identified member of Bcl-2 family, contains a BH2 domain and a putative BH3 domain. It was found to be highly expressed in normal breast tissues, and was associated with favorable prognosis in breast cancer patients. Here, we reported that the mRNA levels of BCL2L12 and its transcript variant BCL2L12A could be upregulated upon cisplatin treatment in MDA-MB-231 breast cancer cells. Knockdown of BCL2L12 and BCL2L12A dramatically inhibited cisplatin-induced apoptosis. In contrast, ectopic expressions of each of the proteins promoted cisplatin-induced apoptosis. These results indicated that decreased expressions or loss of BCL2L12 and BCL2L12A may contribute to the cisplatin resistance in breast cancer patients. Furthermore, we found that cisplatin-induced downregulation of β-catenin was partially suppressed in BCL2L12- and BCL2L12A-knocked down MDA-MB-231 cells, which indicated that knockdown of these two proteins may stabilize β-catenin in cisplatin-induced apoptosis. In short, we proposed that BCL2L12 and BCL2L12A may play an important role in cisplatin-induced apoptosis in MDA-MB-231 breast cancer cells.

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11p58 through AR repression. These data suggest that cyclin D3/CDK11p58 signaling is involved in the negative regulation of AR function.

CDK11(p58), a G2/M-specific protein kinase, has been shown to be associated with apoptosis in many cell lines, with largely unknown mechanisms. Our previous study proved that CDK11(p58)-enhanced cycloheximide (CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells. Here we report for the first time that ectopic expression of CDK11(p58) down-regulates Bcl-2 expression and its Ser70, Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells. Overexpression of Bcl-2 counteracts the pro-apoptotic activity of CDK11(p58). Furthermore, we confirm that the kinase activity of CDK11(p58) is essential to the down-regulation of Bcl-2 as well as apoptosis. Taken together, these results demonstrate that CDK11(p58) down-regulates Bcl-2 in pro-apoptosis pathway depending on its kinase activity, which elicits survival signal in hepatocarcinoma cells.

Human Papilloma virus E6-associated protein (E6-AP), which is known as an E3 ubiquitin ligase, mediates ubiquitination and subsequent degradation of a series of cellular proteins. In this paper, we identify here trihydrophobin 1 (TH1), an integral subunit of the human negative transcription elongation factor (NELF) complex, as a novel E6-AP interaction protein and a target of E6-AP-mediated degradation. Overexpression of E6-AP results in degradation of TH1 in a dose-dependent manner, whereas knock-down of endogenous E6-AP elevates the TH1 protein level. TH1 protein turnover is substantially faster, compared to controls, in cells that overexpressed E6-AP. Wild-type E6-AP promotes the ubiquitination of TH1, while a catalytically inactive point mutant of E6-AP abolishes its ubiquitination. Furthermore, in vitro ubiquitination assay also demonstrates that TH1 can be ubiquitinated by E6-AP. The degradation is blocked by treatment with proteasome inhibitor MG132. Herein, we provide strong evidence that TH1 is a specific substrate that is targeted for degradation through E6-AP-catalyzed polyubiquitination.

beta1,4-Galactosyltransferase V (beta1,4GalT V; EC 2.4.1.38) is considered to be very important in glioma for expressing transformation-related highly branched N-glycans. Recently, we have characterized beta1,4GalT V as a positive growth regulator in several glioma cell lines. However, the role of beta1,4GalT V in glioma therapy has not been clearly reported. In this study, interfering with the expression of beta1,4GalT V by its antisense cDNA in SHG44 human glioma cells markedly promoted apoptosis induced by etoposide and the activation of caspases as well as processing of Bid and expression of Bax and Bak. Conversely, the ectopic expression of beta1,4GalT V attenuated the apoptotic effect of etoposide on SHG44 cells. In addition, both the beta1,4GalT V transcription and the binding of total or membrane glycoprotein with Ricinus communis agglutinin-I (RCA-I) were partially reduced in etoposide-treated SHG44 cells, correlated well with a decreased level of Sp1 that has been identified as an activator of beta1,4GalT V transcription. Collectively, our results suggest that the down-regulation of beta1,4GalT V expression plays an important role in etoposide-induced apoptosis and could be mediated by a decreasing level of Sp1 in SHG44 cells, indicating that inhibitors of beta1,4GalT V may enhance the therapeutic efficiency of etoposide for malignant glioma.

In this study, we reported that small glutamine-rich TPR-containing protein (SGT) interacted with not only Hsp90alpha but also Hsp90beta. Confocal analysis showed that treatment of cells with Hsp90-specific inhibitor geldanamycin (GA) disrupted the interaction of SGT with Hsp90beta and this contributed to the increase of nuclear localization of SGT in HeLa cells. The increased nuclear localization of SGT was further confirmed by the Western blotting in GA-treated HeLa cells and H1299 cells. In our previous study, SGT was found to be a new pro-apoptotic factor, so we wondered whether the sub-cellular localization of SGT was related with cell apoptosis. By confocal analysis we found that the nuclear import of SGT was significantly increased in STS-induced apoptotic HeLa cells, which implied that the sub-cellular localization of SGT was closely associated with Hsp90beta and apoptosis.

Our previous studies have shown that overexpression of beta1,4-galactosyltransferase1 (beta1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of beta1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface beta1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface beta1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of beta1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface beta1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway.

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1--6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.

Abstract 2496 Combretastatin A-4, a derivative of combretastatin, a natural product of the South African tree Combretum caffrum, has been reported to have anti-angiogenic and anti-tubulin effects in different cancer cell lines. We synthesized 48 novel combretastatin analogs to assess anti-leukemia activity in a panel of 12 leukemia cell lines. We identified an analog, VJ-289 [(Z)-3-(1H-indol-2-yl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile] with robust anti-leukemic activity. VJ-289 showed a dose-dependent toxicity to most of the leukemic cell lines tested. The average LD50 for the 12 different leukemia cell lines was 132 nM (95% CI, 91.8–170.5). Specifically, MV4-11 cells demonstrated the most sensitivity to VJ-289 (LD50 = 66 nM), whereas THP-1 was the most resistant (LD50 = 227 nM). Furthermore, when the activity of VJ-289 was tested, five out of 14 primary AML samples demonstrated resistance to VJ-289 with an LD50 > 300nM. The average LD50 for the sensitive primary AML samples was 64.06 nM (95% CI, 35.36–92.76; N=9). Most importantly, normal CD34+ cord blood cells were significantly less affected by VJ-289 (LD50 > 500 nM). Furthermore, VJ-289 was capable of eliminating AML progenitor/stem cells as determined by phenotypic analysis in 15 primary AML samples, colonies forming ability (N=6) and xenotransplant assays (N=6). Overall, we observed a 90.3% decrease in colony formation after treatment with 150 nM VJ-289 relative to untreated control. In contrast, VJ-289 had less impact on colony forming ability of normal hematopoietic stem/progenitor cells from cord blood cells (66.1% decrease relative to untreated; p=0.013). To investigate the role of VJ-289 in leukemic cell apoptosis, various cell survival signaling pathways were examined. Western blotting and intracellular staining/flow cytometry data showed that caspases, including caspase 3 and 8, were activated alongside the cleavage of PARP in a dose-dependent manner. Caspase activation was observed as early as 4 h after treatment with 100 nM VJ-289. PI3K/AKT, MAPK and NF-κB were decreased upon VJ-289 treatment. Moreover, the degradation of MCL1 and the cleavage of Bcl2, two anti-apoptotic Bcl2 family members, were decreased by VJ-289 in a dose- and time-dependent manner. Interestingly, the acetylation of α-tubulin, which is critical for microtubule stabilization, and is involved in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signaling, was transiently induced by VJ-289 within 2 hours, and was inhibited dramatically after 4 hours. In summary, we have identified a combrestastatin A-4 analog, VJ-289, as a new anti-leukemia agent with the ability to ablate blast, progenitor and stem cell populations via induction of caspase activation and α-tubulin acetylation. Studies are underway to determine what modulates sensitivity to VJ-289 across AML specimens. Disclosures: No relevant conflicts of interest to declare.

BCL2L12 has been found to be associated with favorable prognosis in breast cancer patients while correlated with tumorigenesis of glioblastoma and colon cancer. Here, we report that BCL2L12 and its transcript variant BCL2L12A are degraded through ubiquitin-proteasome system (UPS). Interestingly, the ubiquitinations and degradations of BCL2L12 and BCL2L12A are independent of the internal lysine residues but the first N-terminal residues. In addition, HSP70 was identified to interact with BCL2L12 and BCL2L12A and protected them from ubiquitinations and degradations in mammalian cells. In summary, HSP70 protects BCL2L12 and BCL2L12A from N-terminal ubiquitination-mediated proteasomal degradation.Structured summaryMINT-7026352, MINT-7026366: BCL2L12 (uniprotkb:Q9HB09-1) physically interacts (MI:0218) with Hsp70 (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7026290: BCL2L12 (uniprotkb:Q9HB09-1) physically interacts (MI:0218) with ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7026326: Hsp70 (uniprotkb:P08107) physically interacts (MI:0218) with BCL2L12A (uniprotkb:Q9HB09-2) by anti bait coimmunoprecipitation (MI:0006)MINT-7026338: BCL2L12 (uniprotkb:Q9HB09-1) physically interacts (MI:0218) with Hsp70 (uniprotkb:P08107) by anti tag coimmunoprecipitation (MI:0007)MINT-7026304: BCL2L12A (uniprotkb:Q9HB09-2) physically interacts (MI:0218) with Hsp70 (uniprotkb:P08107) by anti tag coimmunoprecipitation (MI:0007)