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Lung cancer is the most lethal cancer globally, accounting for 1.8 million deaths in 2018. Presently, there is a need for novel targets for the development of improved therapeutics. Data mining through genetic screens suggested that Cell Division Cycle 40 (CDC40) is essential for lung cancer cell survival. CDC40 is a WD repeat (WDR) protein that acts as a scaffold mediating protein-protein interactions (PPIs) within the spliceosome complex, and is expected to participate in pre-mRNA splicing pathways. Splicing is a fundamental cellular process for transcriptome and proteome diversity that could support carcinogenesis when hijacked. The therapeutic modulation of cancer-related splicing patterns may thus be an effective strategy for lung cancer treatment, although the precise role of CDC40 in splicing and consequences of CDC40 inhibition remain unknown. With previous success in developing small molecule inhibitors for WDR proteins, our interest now extends to CDC40.Hypothesis: Inhibition of CDC40 will disrupt pre-mRNA splicing and cell cycle control mechanisms in lung cancer cells leading to cell cycle arrest and apoptosis.Through a doxycycline-inducible lentivirus system, several short hairpin RNAs (shRNAs) were introduced into lung cancer cell lines. With CDC40 knockdown (KD), the dependency of 6 lung cancer lines to CDC40 was validated, characterized by significant growth suppression, cell cycle arrest and apoptosis phenotypes. Kinetically, CDC40 mRNA and protein KD were observed as soon as 24h after shRNA induction, followed by cell cycle arrest. This in turn led to Caspase 3/7 activation and an anti-proliferative phenotype. The above phenotypes were rescued upon CDC40 overexpression.RNA-sequencing of a shCDC40-induced lung cancer line revealed the downregulation of genes from cell proliferation and cell structure-related pathways, upregulation of genes from transcription and splicing pathways, as well as a significant increase in intron retention events. RT-qPCR confirmed the downregulation of genes involved in proliferation and glycolipid synthesis, and upregulation of genes involved in splicing. These results indicate that CDC40 plays a role in RNA splicing, and suggest that alterations in splicing through CDC40 inhibition could lead to lung cancer cell growth suppression.Preliminary in silico analysis of CDC40 PPIs identified the top central pocket of the CDC40 WD domain as the most therapeutically tractable site. This region is theorized to interact with CDC5, another spliceosome component. Further investigation of pharmacological (chemical probes) and genetic (point mutation) interventions of CDC40-CDC5 interactions will improve understanding of the role of CDC40 within the spliceosome and the effects of CDC40 PPIs on splicing.Our results demonstrate that CDC40 is essential for lung cancer cell survival, and points to its further investigation as a therapeutic target for lung cancer. Citation Format: Die Hu, Brigitte L. Thériault, Vida Talebian, Julie Owen, Richard Marcellus, Rima Al-awar. Validating the WD repeat protein CDC40 as a potential therapeutic target for lung cancer [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 1222.

Objective: Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy. Post-platinum-based chemotherapy, rapid relapse of chemoresistant tumor is commonly seen. Unidentifiable causative mutations and from reversible nature of acquired chemoresistance, involvement of epigenetic switch regulating SCLC progression is evident. Aim of this screen is to identify epigenetic modifiers that could sensitize SCLC cells to ionizing radiation (IR). Methods: To recapitulate molecular diversity of patients, a range of SCLC cell lines (n=10) from classic or variant and ASCL1 or NeuroD1 sub-groups, driven by varying MYC family amplifications were picked. Short term viability experiment was setup in 3 groups: (a) Epigenetic monotherapy: cells were screened with 10 epigenetic modifiers (0 to 10 µM). (b) IR monotherapy: cells were irradiated from 0 to 8 gy. (c) Combination therapy: cells were treated with different doses of epigenetic probes and IR. Radiosensitization was measured as dose modifying factor at SF63 for each epigenetic modulator. Results: (a) Epigenetic monotherapy: All 10 cell lines were responsive to GSK-J4 (KDMi, IC50 3.1-0.9 µM), SAHA (HDACi, IC50 2.9-0.79 µM) and JIB04 (Jumonji KDMi, IC50 74-2.4 nM). Other interesting epi-probe to which 9/10 cell lines responded was JQ1 (BET-BRDi, IC50 5.7 to 0.15 µM, except SBC-5). Compounds with lesser universal potency were MS023 (PRMTi), UNC0642 (G9a/GLPi) and UNC1999 (EZH2i). All cell lines were irresponsive to PFI3 (SMARCA2/4 BRDi), BAY598 (SMYD2 MTi) and OICR-9429 (WDR5i). (b) IR monotherapy: All 10 cell lines demonstrated different sensitivities to 4 days of IR. They’re ranked in descending order of radiosensitivity as H446, H82, SHP77, LX22, H889, H1092, H196, H69, SBC5, H526. (c) Epi-probes + IR combination therapy: Epi-probes ranked in descending order of number of cell lines they radiosensitized are JQ1, JIB-04, GSK-J4, SAHA, UNC0642, BAY598, MS023, UNC1999, OICR9429 and PFI3. Interestingly, drugs that demonstrated potency as a single agent did not necessarily radiosensitize the cell lines. On the contrary, certain epi-probes that were ineffective as monotherapy radiosensitized a few of the cell lines. H82 is a sensitive cell line which was radiosensitized by all epi-probes used. Conclusions: This screen shows that treating cells with IR in conjunction with epigenetic modifiers may potentially improve therapeutic efficacy of radiotherapy in SCLC. Further validation is being done with long term colonogenic assays followed by in vivo studies for the potent radiosensitizing candidate drugs. It would be therapeutically relevant to correlate the probability of radiosensitization by an epi-probe with the molecular profile of the patients for more predictive targeted therapeutics. Citation Format: Mansi K. Aparnathi, Lifang Song, Ratheesh Subramaniam, Richard Marcellus, Rima Al-awar, Benjamin H. Lok. A screen for epigenetic radiosensitizers in small cell lung 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 3925.

The human adenovirus E4orf4 protein is toxic in both human tumor cells and Saccharomyces cerevisiae . Previous studies indicated that most of this toxicity is dependent on an interaction of E4orf4 protein with the B55 class of regulatory subunits of protein phosphatase 2A (PP2A) and in yeast with the B55 homolog Cdc55. We have found previously that E4orf4 inhibits PP2A activity against at least some substrates. In an attempt to understand the mechanism of this inhibition, we used a genetic approach to identify residues in the seven-bladed β-propeller proteins B55α and Cdc55 required for E4orf4 binding. In both cases, amino-terminal polypeptides composed only of blade 1 and at least part of blade 2 were found to bind E4orf4 and overexpression blocked E4orf4 toxicity in yeast. Furthermore, certain amino acid substitutions in blades 1 and 2 within full-length B55α and Cdc55 resulted in loss of E4orf4 binding. Recent mutational analysis has suggested that segments of blades 1 and 2 present on the top face of B55α form part of the “substrate-binding groove.” Additionally, these segments are in close proximity to the catalytic C subunit of the PP2A holoenzyme. Thus, our results are consistent with the hypothesis that E4orf4 binding could affect the access of substrates, resulting in the failure to dephosphorylate some PP2A substrates.

SELEX was used to create an RNA aptamer targeted to protein tyrosine phosphatase 1B (PTP1B), an enzyme implicated in type 2 diabetes, breast cancer and obesity. We found an aptamer that strongly inhibits PTP1B in vitro with a Ki of less than 600 pM. This slow-binding, high-affinity inhibitor is also highly selective, with no detectable effect on most other tested phosphatases and approximately 300:1 selectivity over the closely related TC-PTP. Through controlled synthesis of truncated variants of the aptamer, we isolated shorter forms that inhibit PTP1B. We also investigated various single-nucleotide modifications to probe their effects on the aptamer's secondary structure and inhibition properties. This family of aptamers represents an exciting option for the development of lead nucleotide-based compounds in combating several human cancers and metabolic diseases.

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70 S6K , were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I 1 PP2A enhanced E4orf4-induced cell killing and G 2 /M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.

SummaryThe “BH3-only” proapoptotic BCL-2 family members are sentinels of intracellular damage. Here, we demonstrated that the BH3-only BID protein partially localizes to the nucleus in healthy cells, is important for apoptosis induced by DNA damage, and is phosphorylated following induction of double-strand breaks in DNA. We also found that BID phosphorylation is mediated by the ATM kinase and occurs in mouse BID on two ATM consensus sites. Interestingly, BID−/− cells failed to accumulate in the S phase of the cell cycle following treatment with the topoisomerase II poison etoposide; reintroducing wild-type BID restored accumulation. In contrast, introducing a nonphosphorylatable BID mutant did not restore accumulation in the S phase and resulted in an increase in cellular sensitivity to etoposide-induced apoptosis. These results implicate BID as an ATM effector and raise the possibility that proapoptotic BID may also play a prosurvival role important for S phase arrest.

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731–6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8–induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

A DNA microarray analysis identified the BH3-only BCL-2 family member, BIK/NBK, as a transcript that is upregulated during induction of apoptosis by oncogenic E1A. E1A depended on wild-type p53 to induce BIK and activate the death program. Further, p53 independently induced BIK RNA and protein, and BIK alone stimulated cell death in p53-null cells, dependent on the activation of caspases. BIK function, however, was abrogated by a disabling point mutation within the BH3 domain. Collectively, these results argue that BIK is a downstream apoptotic effector of p53 in response to a physiological p53-mediated death stimulus provided by E1A. Elevated BCL-2 functioned downstream of p53 and BIK induction to inhibit the E1A death pathway, with the ratio of anti-apoptotic BCL-2 and pro-apoptotic BIK determining cell death or survival in E1A-expressing cells. Cells expressing BCL-2 or treated with the pan caspase inhibitor, zVAD-fmk, allowed accumulation of high levels of cytotoxic BIK compared to control cells. Of note, a significant fraction of either ectopic or endogenous BIK was found associated with the endoplasmic reticulum, suggesting that this organelle, in addition to mitochondria, may be a target of BIK function.

Growth arrest and cell cycle progression are regulated by the retinoblastoma tumour suppressor pRB and related proteins p130 and p107 that bind to and inhibit the E2F family of transcription factors. Although the precise mechanism of this inhibition remains to be established, previous studies indicated the presence of transcriptional repression activity in the 'pocket' of RB family members. We show here that RBP1, a known pRB pocket-binding protein, possesses transcriptional repression activity and associates with p130-E2F and pRB-E2F complexes specifically during growth arrest. Overexpression of RBP1 both inhibited E2F-dependent gene expression and suppressed cell growth. Thus repression of E2F-dependent transcription by RBP1 via RB family members may play a central role in inducing growth arrest.

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.

In the absence of E1B, the 289-amino acid product of human adenovirus type 5 13S E1A induces p53-independent apoptosis by a mechanism that requires viral E4 gene products (Marcellus, R.C., J.C. Teodoro, T. Wu, D.E. Brough, G. Ketner, G.C. Shore, and P.E. Branton. 1996. J. Virol. 70:6207–6215) and involves a mechanism that includes activation of caspases (Boulakia, C.A., G. Chen, F.W. Ng, J.G. Teodoro, P.E. Branton, D.W. Nicholson, G.G. Poirier, and G.C. Shore. 1996. Oncogene. 12:529–535). Here, we show that one of the E4 products, E4orf4, is highly toxic upon expression in rodent cells regardless of the p53 status, and that this cytotoxicity is significantly overcome by coexpression with either Bcl-2 or Bcl-Xl. Conditional expression of E4orf4 induces a cell death process that is characterized by apoptotic hallmark features, such as externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytoplasmic vacuolation, condensation of chromatin, and internucleosomal DNA degradation. However, the wide-spectrum inhibitor of caspases, tetrapeptide zVAD-fmk, does not affect any of these apoptogenic manifestations, and does not alter the kinetics of E4orf4-induced cell death. Moreover, E4orf4 expression does not result in activation of the downstream effector caspase common to most apoptosis-inducing events, caspase-3 (CPP32). We conclude, therefore, that in the absence of E1A, E4orf4 is sufficient by itself to trigger a p53-independent apoptosis pathway that may operate independently of the known zVAD-inhibitable caspases, and that may involve an as yet uncharacterized mechanism.

The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.

The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that no cleavage products appeared in cells expressing ncBID. ncBID was as effective as wtBID in inducing cytochrome c release, caspase activation, and apoptosis. ncBID and wtBID (nc/wtBID) were much less effective than tBID in localizing to mitochondria and in inducing cytochrome c release, but only slightly less effective in inducing apoptosis. Studies with Apaf-1- and caspase-9-deficient primary MEFs indicated that both proteins were essential for nc/wtBID and for tBID-induced apoptosis. Most importantly, expression of non-apoptotic levels of either ncBID or wtBID in Bid -/- MEFs induced a similar and significant enhancement in apoptosis in response to a variety of death signals, which was accompanied by enhanced localization of BID to mitochondria and cytochrome c release. Thus, these results implicate full-length BID as an active player in the mitochondria during apoptosis.

The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD(+) biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD(+) turnover, which makes NAD(+) modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD(+) and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD(+) repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.

The human adenovirus E4orf4 protein, when expressed alone, induces p53-independent death in a wide range of cancer cells. Earlier studies by our groups suggested that although in some cases cell death can be associated with some hallmarks of apoptosis, it is not always affected by caspase inhibitors. Thus it is unlikely that E4orf4-induced cell death occurs uniquely through apoptosis. In the present studies using H1299 human lung carcinoma cells as a model system we found that death is induced in the absence of activation of any of the caspases tested, accumulation of reactive oxygen species, or release of cytochrome c from mitochondria. E4orf4 caused a substantial change in cell morphology, including vigorous membrane blebbing, multiple nuclei in many cells and increased cell volume. Most of these characteristics are not typical of apoptosis, but they are of necrosis. FACS analysis and western blotting for cell cycle markers showed that E4orf4-expressing cells became arrested in G(2)/M and also accumulated high levels of cyclin E. The presence of significant numbers of tetraploid and polyploid cells and some cells with micronuclei suggested that E4orf4 appears to induce death in these cells through a process resulting from mitotic catastrophe.

The secretion of a threshold amount of SAGF can elicit one of the major features of the transformed phenotype, anchorage-independent growth. It appears that, like other cells, NIH 3T3 cells make and secrete SAGF, but not enough to enable them to proliferate in soft agar. The stream of signals from polyoma virus MTAg:pp60c-src:PI-kinase complexes attached to the inner surface of the cell membrane is not enough to raise SAGF secretion to the threshold level for agar growth, but these signals can powerfully enhance the responsiveness to added SAGF. Only when NIH 3T3 cells express all three polyoma T antigens do they secrete enough SAGF and become responsive enough to the factor to form colonies in soft agar. This is in contrast to F111 cells which produce adequate amounts of, and respond to, SAGF after MTAg expression alone. The reasons for this difference remain to be investigated. Obviously, factors expressed during the course of the spontaneous establishment are different for these two lines and they affect the cells' response to MTAg. Clearly, knowing how polyoma virus transforms cells will require a deeper understanding of the deregulation of membrane signaling enzymes by the viral MTAg and the identification and characterization of the autocrine SAGF's that mediate at least part of the neoplastic response to the viral signals.

Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.

Previous studies have indicated that the E4orf4 protein of human adenovirus type 2 (Ad2) induces p53-independent apoptosis. We believe that this process may play a role in cell death and viral spread at the final stages of productive infection. E4orf4 may also be of therapeutic value in treating some diseases, including cancer, through its ability to induce apoptosis when expressed individually. The only previously identified biochemical function of E4orf4 is its ability to associate with the Bα subunit of protein phosphatase 2A (PP2A). We have used a genetic approach to determine the role of such interactions in E4orf4-induced cell death. E4orf4 deletion mutants were of only limited value, as all were highly defective. We found that E4orf4 proteins from most if not all adenovirus serotypes induced cell death, and thus point mutations were introduced that converted the majority of highly conserved residues to alanines. Such mutants were used to correlate Bα-subunit binding, association with PP2A activity, and cell killing following the transfection of appropriate cDNAs into p53-null H1299 or C33A cells. The results indicated that binding of the Bα subunit is essential for induction of cell death, as every mutant that failed to bind efficiently was totally defective for cell killing. This class of mutations (class I) largely involved residues between amino acids 51 and 89. Almost all E4orf4 mutant proteins that associated with PP2A killed cancer cells at high levels; however, several mutants that associated with significant levels of PP2A were defective for killing (class II). Thus, binding of E4orf4 to PP2A is essential for induction of p53-independent apoptosis, but E4orf4 may possess one or more additional functions required for cell killing.

▪ Abstract Successful viral replication requires not only the efficient production and spread of progeny, but also evasion of host defense mechanisms that limit replication by killing infected cells. In addition to inducing immune and inflammatory responses, infection by most viruses triggers apoptosis or programmed cell death of the infected cell. This cell response often results as a compulsory or unavoidable by-product of the action of critical viral replicative functions. In addition, some viruses seem to use apoptosis as a mechanism of cell killing and virus spread. In both cases, successful replication relies on the ability of certain viral products to block or delay apoptosis until sufficient progeny have been produced. Such proteins target a variety of strategic points in the apoptotic pathway. In this review we summarize the great amount of recent information on viruses and apoptosis and offer insights into how this knowledge may be used for future research and novel therapies.

Previous studies by our group showed that infection of human and rodent cells by human adenovirus type 5 (Ad5) results in the induction of p53-independent apoptosis and cell death that are dependent upon transactivation of early region 4 (E4). To identify which E4 products are involved, studies were conducted with p53-deficient human SAOS-2 cells infected with various Ad5 E4 mutants. An E4orf6-deficient mutant was defective in cell killing, whereas another that expressed only E4orf6 and E4orf4 killed like wild-type virus, suggesting that E4orf6 may be responsible for cytotoxicity; however, a mutant expressing only E4orf4 induced high levels of cell death, indicating that this E4 product may also be able to induce cytotoxicity. To define the E4 cell death-inducing functions more precisely, cDNAs encoding individual E4 products were introduced into cells by DNA transfection in the absence of other Ad5 proteins. In cotransfections with a cDNA encoding firefly luciferase, enzymatic activity was high in all cases except with E4orf4, where luciferase levels were less than 20% of those in controls. In addition, drug selection of several cell types following transfection with retroviral vector DNA encoding individual E4 products as well as puromycin resistance yielded a large number of cell colonies except when E4orf4 was expressed. These data demonstrated that E4orf4 is the only E4 product capable of independent cell killing. Cell death induced by E4orf4 was due to apoptosis, as evidenced by 4′,6-diamidino-2-phenylindole (DAPI) staining of cell nuclei in E4orf4-expressing cells. Thus, although E4orf6 may play some role, these results suggested that E4orf4 may be the major E4 product responsible for induction of p53-independent apoptosis.

A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.

In the absence of E1B, the 289- and 243-residue E1A products of human adenovirus type 5 induce p53-dependent apoptosis. However, our group has shown recently that the 289-residue E1A protein is also able to induce apoptosis by a p53-independent mechanism (J. G. Teodoro, G. C. Shore, and P. E. Branton, Oncogene 11:467-474, 1995). Preliminary results suggested that p53-independent cell death required expression of one or more additional adenovirus early gene products. Here we show that both the E1B 19-kDa protein and cellular Bcl-2 inhibit or significantly delay p53-independent apoptosis. Neither early region E2 or E3 appeared to be necessary for such cell death. Analysis of a series of E1A mutants indicated that mutations in the transactivation domain and other regions of E1A correlated with E1A-mediated transactivation of E4 gene expression. Furthermore, p53-deficient human SAOS-2 cells infected with a mutant which expresses E1B but none of the E4 gene products remained viable for considerably longer times than those infected with wild-type adenovirus type 5. In addition, an adenovirus vector lacking both E1 and E4 was unable to induce DNA degradation and cell killing in E1A-expressing cell lines. These data showed that an E4 product is essential for E1A-induced p53-independent apoptosis.

Human adenovirus early region 1A (E1A) proteins act as transcriptional regulators and function in the control of DNA synthesis and cell transformation. Little is known about how these viral products are functionally regulated. E1A proteins of adenovirus serotype 5 (Ad5) are phosphorylated at several serine residues and previous studies had indicated that both Ser-89 and Ser-219 are substrates for one or more of the cdc2 family of cell cycle kinases. A second residue near the amino terminus, Ser-96, may also be a site. Although phosphorylation of Ser-89 causes a major shift in gel mobility, the effect on E1A biological activity is unclear. In the present studies we have shown by mutational analysis that phosphorylation at Ser-89 also regulates phosphorylation at Ser-96, suggesting that the gel mobility shift is the result of multiple phosphorylation events. Phosphorylation at Ser-89 did not seem to affect E1A-mediated repression of the simian virus 40 enhancer or trans-activation of the E3 promoter significantly, but it did appear to have a modest but significant effect on transformation of primary baby rat kidney cells.

Anchorage-dependent, nontumorigenic rat F111 fibroblasts have a low level of membrane-associated protein kinase C (PKC) activity. After expression of the polyoma virus middle tumor antigen this activity increased, the cells grew in agar and formed tumors after injection into syngeneic rats or nude mice. Contrary to F111, a clone of mouse NIH 3T3 fibroblasts has a high membrane-associated PKC activity and is as tumorigenic as polyoma-transformed cells, although this clone is still anchorage-dependent. Therefore, membrane-associated PKC activation might be one of the signals leading to tumorigenicity but not necessarily anchorage-independence.

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36–69 within conserved region 1 (CR1), and residues 121–138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.Key words: DNA tumour viruses, transformation, oncogenes, cell cycle, protein complexes.

Obatoclax (GX15-070), a synthetic small molecule pan-Bcl-2 family inhibitor, is currently under investigation in multiple Phase II trials directed at hematologic and solid tumor malignancies. Inhibition of pro-survival Bcl-2 members antagonizes their ability to neutralize the death effectors Bax and Bak, which are required for obatoclax-induced cellular toxicity. The hydrophobic character of obatoclax is consistent with its predicted occupation of a hydrophobic pocket in the lower region of the Bcl-2 BH3 binding groove. Based on comparisons with the published results of 10 known small molecule inhibitors, obatoclax is computationally predicted to have a significant binding affinity for Bcl-2 (Ki = 220 nanoM). A recent report (Zhai et al., Cell Death Differ. 2006 13: 1419-21), describing the interactions of synthetic BH3 peptides with Bcl-2 family proteins by fluorescence polarization in aqueous medium (PBS), indicated that obatoclax selectively antagonizes such interactions for all pro-survival members tested (Bcl-2, Bcl-Xl, Mcl-1, Bcl-w, A1, and Bcl-b), but did not affect control protein interactions. IC50s for obatoclax ranged from 1 to 7 microM. Under the aqueous conditions of this assay, however, obatoclax is largely insoluble and therefore this report likely underestimates the potency of the compound. In situ, Bcl-2 proteins reside integrated in the hydrophobic membranes of mitochondria and ER. We took advantage of the fact that Mcl-1 constitutively interacts with Bak in the mitochondrial outer membrane, thus presenting the opportunity to investigate the potency of obatoclax in situ on a native Bcl-2 protein-protein interaction. Employing chemical crosslinking to detect Mcl-1/Bak dimers in intact mitochondria, obatoclax was found to disrupt these interactions with an IC50 less than 10 nanoM, similar to that observed for inhibition by synthetic BH3 peptides corresponding to Noxa and Bim. In contrast to Bim peptide, which induced release of cytochrome c from isolated mitochondria, obatoclax did not, indicating that it is a sensitizer rather than an activator of the intrinsic mitochondrial apoptosis program. Bax or Bak are toxic to yeast cells, but are restrained by either Mcl-1, Bcl-2, Bcl-Xl, or Bcl-w. In each case, obatoclax treatment re-instated Bax or Bak toxicity, confirming the pan-inhibitor properties of the compound. Among Bcl-2 proteins, Mcl-1 rapidly turns over at steady state and is subject to ubiquitin-mediated degradation by the 26S proteasome. Not surprisingly, the proteasome inhibitor bortezomib significantly enhances Mcl-1 protein levels in various settings, including mantle cell lymphoma (MCL) cells. Bortezomib also induced Noxa upregulation in MCL, and obatoclax and Noxa appeared to cooperate to displace Bak from Mcl-1. As predicted, obatoclax and bortezomib exhibited synergistic toxicity in MCL lines in vitro and in cells derived from MCL patients ex vivo. Collectively, therefore, a biochemical rationale has been established to interrogate obatoclax-bortezomib combinations in clinical trials directed at mantle cell lymphoma.

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19- and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.