Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells.

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.

Discovery of Small Molecule Bak Activator for Lung Cancer Therapy.

Rationale: Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. Methods: The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. Results: We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Conclusions: Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.

Correction to: Small Molecule KRAS Agonist for Mutant KRAS Cancer Therapy.

An amendment to this paper has been published and can be accessed via the original article.

EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair.

Small cell lung cancer (SCLC) is a highly aggressive malignancy with poor outcomes associated with resistance to cisplatin-based chemotherapy. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 (PRC2), which silences transcription through trimethylation of histone H3 lysine 27 (H3K27me3) and has emerged as an important therapeutic target with inhibitors targeting its methyltransferase activity under clinical investigation. Here, we show that EZH2 has a non-catalytic and PRC2-independent role in stabilizing DDB2 to promote nucleotide excision repair (NER) and govern cisplatin resistance in SCLC. Using a synthetic lethality screen, we identified important regulators of cisplatin resistance in SCLC cells, including EZH2. EZH2 depletion causes cellular cisplatin and UV hypersensitivity in an epistatic manner with DDB1-DDB2. EZH2 complexes with DDB1-DDB2 and promotes DDB2 stability by impairing its ubiquitination independent of methyltransferase activity or PRC2, thereby facilitating DDB2 localization to cyclobutane pyrimidine dimer crosslinks to govern their repair. Furthermore, targeting EZH2 for depletion with DZNep strongly sensitizes SCLC cells and tumors to cisplatin. Our findings reveal a non-catalytic and PRC2-independent function for EZH2 in promoting NER through DDB2 stabilization, suggesting a rationale for targeting EZH2 beyond its catalytic activity for overcoming cisplatin resistance in SCLC.

Bcl2-induced DNA replication stress promotes lung carcinogenesis in response to space radiation.

Space radiation is characterized by high-linear energy transfer (LET) ionizing radiation. The relationships between the early biological effects of space radiation and the probability of cancer in humans are poorly understood. Bcl2 not only functions as a potent antiapoptotic molecule but also as an oncogenic protein that induces DNA replication stress. To test the role and mechanism of Bcl2 in high-LET space radiation-induced lung carcinogenesis, we created lung-targeting Bcl2 transgenic C57BL/6 mice using the CC10 promoter to drive Bcl2 expression selectively in lung tissues. Intriguingly, lung-targeting transgenic Bcl2 inhibits ribonucleotide reductase activity, reduces dNTP pool size and retards DNA replication fork progression in mouse bronchial epithelial cells. After exposure of mice to space radiation derived from 56iron, 28silicon or protons, the incidence of lung cancer was significantly higher in lung-targeting Bcl2 transgenic mice than in wild-type mice, indicating that Bcl2-induced DNA replication stress promotes lung carcinogenesis in response to space radiation. The findings provide some evidence for the relative effectiveness of space radiation and Bcl-2 at inducing lung cancer in mice.

Mcl-1 Interacts with Akt to Promote Lung Cancer Progression.

Mcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non-small cell lung cancer. Using the Mcl-1-binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer. SIGNIFICANCE: These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.

Small Molecule KRAS Agonist for Mutant KRAS Cancer Therapy.

BACKGROUND: Lung cancer patients with KRAS mutation(s) have a poor prognosis due in part to the development of resistance to currently available therapeutic interventions. Development of a new class of anticancer agents that directly targets KRAS may provide a more attractive option for the treatment of KRAS-mutant lung cancer. RESULTS: Here we identified a small molecule KRAS agonist, KRA-533, that binds the GTP/GDP-binding pocket of KRAS. In vitro GDP/GTP exchange assay reveals that KRA-533 activates KRAS by preventing the cleavage of GTP into GDP, leading to the accumulation of GTP-KRAS, an active form of KRAS. Treatment of human lung cancer cells with KRA-533 resulted in increased KRAS activity and suppression of cell growth. Lung cancer cell lines with KRAS mutation were relatively more sensitive to KRA-533 than cell lines without KRAS mutation. Mutating one of the hydrogen-bonds among the KRA-533 binding amino acids in KRAS (mutant K117A) resulted in failure of KRAS to bind KRA-533. KRA-533 had no effect on the activity of K117A mutant KRAS, suggesting that KRA-533 binding to K117 is required for KRA-533 to enhance KRAS activity. Intriguingly, KRA-533-mediated KRAS activation not only promoted apoptosis but also autophagic cell death. In mutant KRAS lung cancer xenografts and genetically engineered mutant KRAS-driven lung cancer models, KRA-533 suppressed malignant growth without significant toxicity to normal tissues. CONCLUSIONS: The development of this KRAS agonist as a new class of anticancer drug offers a potentially effective strategy for the treatment of lung cancer with KRAS mutation and/or mutant KRAS-driven lung cancer.

Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy.

DNA double-strand breaks (DSBs) are mainly repaired either by homologous recombination (HR) or by nonhomologous end-joining (NHEJ) pathways. Here, we showed that myeloid cell leukemia sequence 1 (Mcl-1) acts as a functional switch in selecting between HR and NHEJ pathways. Mcl-1 was cell cycle-regulated during HR, with its expression peaking in S/G2 phase. While endogenous Mcl-1 depletion reduced HR and enhanced NHEJ, Mcl-1 overexpression resulted in a net increase in HR over NHEJ. Mcl-1 directly interacted with the dimeric Ku protein complex via its Bcl-2 homology 1 and 3 (BH1 and BH3) domains, which are required for Mcl-1 to inhibit Ku-mediated NHEJ. Mcl-1 also promoted DNA resection mediated by the Mre11 complex and HR-dependent DSB repair. Using the Mcl-1 BH1 domain as a docking site, we identified a small molecule, MI-223, that directly bound to BH1 and blocked Mcl-1-stimulated HR DNA repair, leading to sensitization of cancer cells to hydroxyurea- or olaparib-induced DNA replication stress. Combined treatment with MI-223 and hydroxyurea or olaparib exhibited a strong synergy against lung cancer in vivo. This mechanism-driven combination of agents provides a highly attractive therapeutic strategy to improve lung cancer outcomes.

Modulation of Bax and mTOR for Cancer Therapeutics.

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001-12. ©2017 AACR.

Targeting DNA Replication Stress for Cancer Therapy.

The human cellular genome is under constant stress from extrinsic and intrinsic factors, which can lead to DNA damage and defective replication. In normal cells, DNA damage response (DDR) mediated by various checkpoints will either activate the DNA repair system or induce cellular apoptosis/senescence, therefore maintaining overall genomic integrity. Cancer cells, however, due to constitutive growth signaling and defective DDR, may exhibit "replication stress" -a phenomenon unique to cancer cells that is described as the perturbation of error-free DNA replication and slow-down of DNA synthesis. Although replication stress has been proven to induce genomic instability and tumorigenesis, recent studies have counterintuitively shown that enhancing replicative stress through further loosening of the remaining checkpoints in cancer cells to induce their catastrophic failure of proliferation may provide an alternative therapeutic approach. In this review, we discuss the rationale to enhance replicative stress in cancer cells, past approaches using traditional radiation and chemotherapy, and emerging approaches targeting the signaling cascades induced by DNA damage. We also summarize current clinical trials exploring these strategies and propose future research directions including the use of combination therapies, and the identification of potential new targets and biomarkers to track and predict treatment responses to targeting DNA replication stress.

BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth.

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy and remains incurable. B-cell lymphoma-2 (BCL2) protein correlates with the survival and the drug resistance of myeloma cells. BH3 mimetics have been developed to disrupt the binding between BCL2 and its pro-apoptotic BCL2 family partners for the treatment of MM, but with limited therapeutic efficacy. We recently identified a small molecule BDA-366 as a BCL2 BH4 domain antagonist, converting it from an anti-apoptotic into a pro-apoptotic molecule. In this study, we demonstrated that BDA-366 induces robust apoptosis in MM cell lines and primary MM cells by inducing BCL2 conformational change. Delivery of BDA-366 substantially suppressed the growth of human MM xenografts in NOD-scid/IL2Rγnull mice, without significant cytotoxic effects on normal hematopoietic cells or body weight. Thus, BDA-366 functions as a novel BH4-based BCL2 inhibitor and offers an entirely new tool for MM therapy.

The role of adiponectin in obesity-associated female-specific carcinogenesis.

Adipose tissue is a highly vascularized endocrine organ, and its secretion profiles may vary with obesity. Adiponectin is secreted by adipocytes that make up adipose tissue. Worldwide, obesity has been designated a serious health problem among women and is associated with a variety of metabolic disorders and an increased risk of developing cancer of the cervix, ovaries, uterus (uterine/endometrial), and breast. In this review, the potential link between obesity and female-specific malignancies is comprehensively presented by discussing significant features of the intriguing and complex molecule, adiponectin, with a focus on recent findings highlighting its molecular mechanism of action in female-specific carcinogenesis.

Establishment of Patient-derived Xenografts in Mice.

Patient-derived xenograft (PDX) models for cancer research have recently attracted considerable attention in both the academy and industry (Hidalgo et al., 2014; Wilding and Bodmer, 2014). PDX models have been developed from different tumor types including lung cancer to improve the drug development process. These models are used for pre-clinical drug evaluation and can be used for the predictive results of clinical outcomes because they conserve original tumor characteristics such as heterogeneity, complexity and molecular diversity (Kopetz et al., 2012). Additionally, PDX model provides the potential tool for the personalized drug therapy. In this protocol, we present methods for the establishment of PDX in mice using primary tumor tissues from patients with small cell lung cancer (SCLC).

Targeting KRAS-mutant non-small cell lung cancer: challenges and opportunities.

Oncogenic mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) occur in 15%-30% of non-small cell lung cancer (NSCLC). However, despite decades of intensive research, there is still no direct KRAS inhibitor with clinically proven efficacy. Considering its association with poor treatment response and prognosis of lung cancer, developing an effective inhibitory approach is urgently needed. Here, we review different strategies currently being explored to target KRAS-mutant NSCLC, discuss opportunities and challenges, and also propose some novel methods and concepts with the promise of clinical application.

Small-Molecule Bcl2 BH4 Antagonist for Lung Cancer Therapy.

The BH4 domain of Bcl2 is required for its antiapoptotic function, thus constituting a promising anticancer target. We identified a small-molecule Bcl2-BH4 domain antagonist, BDA-366, that binds BH4 with high affinity and selectivity. BDA-366-Bcl2 binding induces conformational change in Bcl2 that abrogates its antiapoptotic function, converting it from a survival molecule to a cell death inducer. BDA-366 suppresses growth of lung cancer xenografts derived from cell lines and patient without significant normal tissue toxicity at effective doses. mTOR inhibition upregulates Bcl2 in lung cancer cells and tumor tissues from clinical trial patients. Combined BDA-366 and RAD001 treatment exhibits strong synergy against lung cancer in vivo. Development of this Bcl2-BH4 antagonist may provide a strategy to improve lung cancer outcome.

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation.

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. (56)iron and (28)silicon) by inhibiting Mre11 complex activity. Exposure of cells to (56)iron or (28)silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.

Small-molecule Bax agonists for cancer therapy.

Bax, a central death regulator, is required at the decisional stage of apoptosis. We recently identified serine 184 (S184) of Bax as a critical functional switch controlling its proapoptotic activity. Here we used the structural pocket around S184 as a docking site to screen the NCI library of small molecules using the UCSF-DOCK programme suite. Three compounds, small-molecule Bax agonists SMBA1, SMBA2 and SMBA3, induce conformational changes in Bax by blocking S184 phosphorylation, facilitating Bax insertion into mitochondrial membranes and forming Bax oligomers. The latter leads to cytochrome c release and apoptosis in human lung cancer cells, which occurs in a Bax- but not Bak-dependent fashion. SMBA1 potently suppresses lung tumour growth via apoptosis by selectively activating Bax in vivo without significant normal tissue toxicity. Development of Bax agonists as a new class of anticancer drugs offers a strategy for the treatment of lung cancer and other Bax-expressing malignancies.

Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing.

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.