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LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.

Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M., (©2016 American Association for Cancer Research.)

Oncogene addiction due to Myc deregulation has been identified in a variety of tumor types. In order to identify pharmacological agents that cause selective apoptosis in tumors with deregulated Myc expression, we designed a cell-based screening assay based on our Anti-cancer Screening Apoptosis Program (ASAP) technology targeting increased activity in a "Myc-addicted" cancer cell panel. We have identified a novel set of substituted 4-aryl-3-(3-aryl-1-oxo-2-propenyl)-2(1H)-quinolinones that activates apoptosis in cancer cell lines with deregulated Myc, but show low activity in cell lines where Myc is not deregulated. Apoptosis induced by these compounds is rapid, and is associated with a significant downregulation of Myc protein. Selective knockdown of Myc levels in these cells by RNA interference increased sensitivity to apoptosis with compound treatment. By targeting the Myc pathway in Myc-addicted cancer cells, we have identified a novel class of apoptotic inducers that selectively and efficiently target cancer cells with deregulated Myc.

Cultured primary human cells have been widely used to assess the selectivity of oncolytic viruses as potential anticancer agents. As culture conditions can potentially have a significant impact on virus replication and ultimately cell killing, we evaluated the effects of dl309, a wild-type adenovirus, and dl01 / 07, a conditionally replicating adenovirus mutant, on quiescent and proliferating primary mammary epithelial cells. When primary cells were induced into quiescence, both viruses exhibited similar attenuated cell killing. However, cell killing by dl309 was superior to dl01 / 07 in proliferating primary cells. Analysis of viral effects at the level of entry, E2F activation, DNA replication, and late gene expression indicated that attenuation of dl309 in quiescent cells correlated with decreased expression of viral late genes such as hexon. In contrast, attenuation of dl01 / 07 in quiescent cells correlated with inefficient induction of E2F activity and inability to undergo efficient DNA replication. In proliferating cells, dl309 replicated efficiently, whereas dl01 / 07 still showed attenuated replication. In summary, our results indicate the intrinsic preference of wild-type adenoviruses for killing proliferating cells, which is an attractive feature for using adenoviruses as oncolytic agents. These results also highlight the need for the use of appropriate growth conditions for primary cells in vitro to distinguish subtle differences in cell killing among various oncolytic viruses.

In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.

TP53 is the most commonly altered tumor-suppressor gene in cancer and is currently being tested in Phase II/III gene replacement trials. Many tumors contain wild-type TP53 sequence with elevated MDM2 protein levels, targeting p53 for degradation. These tumors are more refractory to treatment with exogenous wild-type p53. Here we generate a recombinant adenovirus expressing a p53 variant, rAd-p53 (d 13-19), that is deleted for the amino acid sequence necessary for MDM2 binding (amino acids 13-19). We compared the apoptotic activity of rAd-p53 (d 13-19) with that of a recombinant adenovirus expressing wild-type p53 (rAd-p53) in cell lines that differ in endogenous p53 status. rAd-p53 (d 13-19) caused higher levels of apoptosis in p53 wild-type tumor lines compared with wild-type p53 treatment, as measured by annexin V-FITC staining. In p53-altered tumor lines, rAd-p53 (d 13-19) showed apoptotic activity similar to that seen with wild-type p53 treatment. In normal cells, no increase in cytopathicity was detected with rAd-p53 (d 13-19) compared with wild-type p53 treatment. This variant protein displayed synergy with chemotherapeutic agents to inhibit proliferation of ovarian and breast cell lines. The p53 variant showed greater antitumor activity in an established p53 wild-type tumor compared with treatment with wild-type p53. The p53 variant represents a means of expanding TP53 gene therapy to tumors that are resistant to p53 treatment due to the cellular responses to wild-type p53.

The oncolytic effect of adenoviruses may provide an efficient means to destroy tumor tissue if viruses could be developed with sufficient selectivity and efficacy. In this report we have characterized several adenoviruses, each with different mutations in the E1 region, for selective cytopathic effect in tumor cells in vitro and for their ability to inhibit tumor growth in vivo. Of the E1 mutants tested, we have identified one, E1Adl01/07, which preferentially induces cytopathic effects in a range of tumor cells versus primary cells. In addition, E1Adl01/07 significantly inhibited tumor growth and increased survival of mice in several models of human cancer. These results suggest that E1Adl01/07 might serve as an effective cancer therapeutic, combining both selectivity and efficacy.

Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.

Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCB-infected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with this recombinant adenoviral vector has a potential use in cancer gene therapy.

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.

We have constructed recombinant human adenoviruses that express wild-type human p53 under the control of either the Ad 2 major late promoter (MLP) or the human cytomegalovirus (CMV) immediate early gene promoter. Each construct replaces the Ad 5 E1a and E1b coding sequences necessary for viral replication with the p53 cDNA and MLP or CMV promoter. These p53/Ad recombinants are able to express p53 protein in a dose-dependent manner in infected human cancer cells. Tumor suppressor activity of the expressed p53 protein was assayed by several methods. [3H]Thymidine incorporation assays showed that the recombinant adenoviruses were capable of inhibiting DNA synthesis in a p53-specific, dose-dependent fashion. Ex vivo treatment of Saos-2 tumor cells, followed by injection of the treated cells into nude mice, led to complete tumor suppression using the MLP/p53 recombinant. Following a single injection of CMV/p53 recombinant adenovirus into the peritumoral space surrounding an in vivo established tumor derived from a human small cell lung carcinoma cell line (NIH-H69), we were able to detect p53 mRNA in the tumors at 2 and 7 days post-injection. Continued treatment of established H69 tumors with MLP/p53 recombinant led to reduced tumor growth and increased survival time compared to control treated animals. These results indicate that recombinant adenoviruses expressing wild-type p53 may be useful vectors for gene therapy of human cancer.