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Despite advances in targeted therapy, lung cancer remains the most common cause of cancer-related mortality in the United States. Chromosomal instability is a prominent feature in lung cancer and, because it rarely occurs in normal cells, it represents a potential therapeutic target. Our prior work discovered that lung cancer cells undergo anaphase catastrophe in response to inhibition of cyclin-dependent kinase 2 (CDK2), followed by apoptosis and reduced growth. In this study, the effects and mechanisms of the multi-CDK inhibitor dinaciclib on lung cancer cells were investigated. We sought to determine the specificity of CDK-dependent induction of anaphase catastrophe. Live cell imaging provided direct evidence that dinaciclib caused multipolar cell divisions resulting in extensive chromosome missegregation. Genetic knockdown of dinaciclib CDK targets revealed that repression of CDK2 and CDK1, but not CDK5 or CDK9, triggered anaphase catastrophe in lung cancer cells. Overexpression of CP110, which is a mediator of CDK2 inhibitor–induced anaphase catastrophe (and a CDK1 and 2 phosphorylation substrate), antagonized anaphase catastrophe and apoptosis following dinaciclib treatment. Consistent with our previous findings, acquisition of activated KRAS sensitized lung cancer cells to dinaciclib-mediated anaphase catastrophe and cell death. Combining dinaciclib with the mitotic inhibitor taxol augmented anaphase catastrophe induction and reduced cell viability of lung cancer cells. Thus, the multi-CDK inhibitor dinaciclib causes anaphase catastrophe in lung cancer cells and should be investigated as a potential therapeutic for wild-type and KRAS-mutant lung cancer, individually or in combination with taxanes. Mol Cancer Ther; 15(11); 2758–66. ©2016 AACR.

KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo. To solidify this association, loss of Usp18 in KrasLA2/+/Usp18−/− mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas.Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905–14. ©2017 AACR.

Knock-down of Usp33 in murine lung cancer cells does not affect CP110 protein expression. Effects of Usp33 knock-down at 24,48, 72 and 96 hours on CP110 expression in ED-1 cells.

Movie video files of a) H1299 and b) HOP62 cells were treated with 25 or 50 nM dinaciclib and continuously imaged as described in the Methods.

The spindle assembly checkpoint maintains genomic integrity. A key component is tyrosine threonine kinase (TTK, also known as Mps1). TTK antagonism is hypothesized to cause genomic instability and cell death. Interrogating The Cancer Genome Atlas revealed high TTK expression in lung adenocarcinomas and squamous cell cancers versus the normal lung (P < 0.001). This correlated with an unfavorable prognosis in examined lung adenocarcinoma cases (P = 0.007). TTK expression profiles in lung tumors were independently assessed by RNA in situ hybridization. CFI-402257 is a highly selective TTK inhibitor. Its potent antineoplastic effects are reported here against a panel of well-characterized murine and human lung cancer cell lines. Significant antitumorigenic activity followed independent treatments of athymic mice bearing human lung cancer xenografts (6.5 mg/kg, P < 0.05; 8.5 mg/kg, P < 0.01) and immunocompetent mice with syngeneic lung cancers (P < 0.001). CFI-402257 antineoplastic mechanisms were explored. CFI-402257 triggered aneuploidy and apoptotic death of lung cancer cells without changing centrosome number. Reverse phase protein arrays (RPPA) of vehicle versus CFI-402257–treated lung cancers were examined using more than 300 critical growth-regulatory proteins. RPPA bioinformatic analyses discovered CFI-402257 enhanced MAPK signaling, implicating MAPK antagonism in augmenting TTK inhibitory effects. This was independently confirmed using genetic and pharmacologic repression of MAPK that promoted CFI-402257 anticancer actions. TTK antagonism exerted marked antineoplastic effects against lung cancers and MAPK inhibition cooperated. Future work should determine whether CFI-402257 treatment alone or with a MAPK inhibitor is active in the lung cancer clinic.

The siRNA sequences used in this study.

Ubiquitin specific peptidase 18 (USP18), previously known as UBP43, is the IFN-stimulated gene 15 (ISG15) deconjugase. USP18 removes ISG15 from substrate proteins. This study reports that USP18-null mice (vs. wild-type mice) exhibited lower lipolysis rates, altered fat to body weight ratios, and cold sensitivity. USP18 is a regulator of lipid and fatty acid metabolism. Prior work established that USP18 promotes lung tumorigenesis. We sought to learn whether this occurs through altered lipid and fatty acid metabolism. Loss of USP18 repressed adipose triglyceride lipase (ATGL) expression; gain of USP18 expression upregulated ATGL in lung cancer cells. The E1-like ubiquitin activating enzyme promoted ISG15 conjugation of ATGL and destabilization. Immunoprecipitation assays confirmed that ISG15 covalently conjugates to ATGL. Protein expression of thermogenic regulators was examined in brown fat of USP18-null versus wild-type mice. Uncoupling protein 1 (UCP1) was repressed in USP18-null fat. Gain of USP18 expression augmented UCP1 protein via reduced ubiquitination. Gain of UCP1 expression in lung cancer cell lines enhanced cellular proliferation. UCP1 knockdown inhibited proliferation. Beta-hydroxybutyrate colorimetric assays performed after gain of UCP1 expression revealed increased cellular fatty acid beta-oxidation, augmenting fatty acid beta-oxidation in Seahorse assays. Combined USP18, ATGL, and UCP1 profiles were interrogated in The Cancer Genome Atlas. Intriguingly, lung cancers with increased USP18, ATGL, and UCP1 expression had an unfavorable survival. These findings reveal that USP18 is a pharmacologic target that controls fatty acid metabolism.Implications:USP18 is an antineoplastic target that affects lung cancer fatty acid metabolism.

Figure S1: The structure of CFI-402257. Figure S2: TTK mRNA expression profiles in lung cancer cases with different smoking patterns, as shown in panel A. In this left panel N means no smoking history while Y indicates the presence of a smoking history. That was displayed in the right panel as never, former or current smokers. Panel B displays the anti-proliferative effects of CFI-402257 relative to vehicle treatments in lung adenocarcinoma cell lines. The area under the curve was calculated based on the proportion of control values and log10 dosage values to generate dose-response curves. Error bars are standard deviations. Figure S3: Immunoblot analysis of ED1 (A) and A549 (B) lung cancer cells after independent transfection with TTK-targeting (siRNA1 and siRNA2) or control siRNAs. Figure S4: Representative photomicrographs of 393P (A) or H1299 (B) lung cancer cell line-derived tumors after 21 days of CFI-402257 or vehicle treatments. (C) Syngeneic 393P tumor-bearing mice were treated with vehicle, 8.5 mg/kg CFI-402257 orally for 5 days, after which tumors were harvested for RPPA analyses (n = 5 mice per group). Comparison of the 8.5mg/kg CFI-402257-treated group to vehicle-treated groups. The proteins displayed above the red line are significantly different (P < 0.05). Figure S5: (A) The association between survival and expression of MAP2K1/MAP2K2 and TTK in combination using TCGA. Green: MAP2K low/TTK low, purple: MAP2K low/TTK high, orange: MAP2K high/TTK low and red: MAP2K high/TTK high. (B) The combination index (CI) for each dosage pair was calculated. The dash line indicates CI=1.0. Figure S6: TTK mutations in lung adenocarcinomas (A) and squamous cell carcinomas (B) in TCGA database.

Supplementary table

Contains supplementary methods and supplementary figure legends.

Movie video files of HOP62 cells as described in the Methods.

S1. Introduction of enzymatically-active USP18 overcomes growth inhibition of USP18-repressed lung cancer cells. S2. Loss of USP18 expression reduces growth of KRAS mutant human lung cancer cells. S3. Interferon (IFN) treatment of lung cancer cells having repressed USP18 expression promotes growth inhibition. S4. Modifying USP18 expression alters KRAS protein levels. S5. USP18 regulates KRAS post-transcriptionally. S6. Repression of USP18 expression downregulates AKT and MAPK signaling pathways. S7. KRAS is ISG15-modified. S8. USP18 knockdown promotes KRAS localization to the endomembrane compartment. S9. KrasLA2/+ mice were crossed with Usp18-/- mice to generate KrasLA2/+/Usp18-/- compound mice. S10. KRAS mRNA is increased with presence of activating KRAS mutations and USP18 protein expression is augmented in the presence of high KRAS levels.

S1. USP18 regulates metabolism-related proteins by Reverse Phase Protein Array (RPPA) in A549 lung cancer cells. S2. This figure compares CellTiter-Glo assays and manual counting for lung cancer cell proliferation at day 3. S3. The mRNA expression profiles of USP18, ATGL and UCP1 in the indicated different cancer types using TCGA database. S4. Combined USP18, ATGL and UCP1 expression profiles in cancers that were using TCGA database. S5. USP18 regulates lipid metabolism in lung cancer cell lines. S6. Differential ATGL expression profiles by immunohistochemical staining of human lung cancer cases using the Human Protein Atlas.

Supplementary figures

Supplemental File S1. The siRNA sequences and real-time qPCR primer sequences.

Supplemental File S2. A representative video of a Hop62 cell that undergoes multipolar cell division.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 2029–38. ©2015 AACR.

Supplemental Fig. S3. (A) Schematic diagram showing the relationship between CDK2, CP110, activated KRAS and anaphase catastrophe. (B) Engineered overexpression of CP110 was detected in A549, Hop62, H522 and H460 cells (versus control transfectants) using anti-HA antibody after 24 and 48 hours of transfection. A representative immunoblot for each cell line is displayed.

Supplemental Fig. S5. Representative CP110 immunohistochemistry staining of lung normal (N) versus malignant (T) tissues harvested independently from KRAS wild-type transgenic mice and KRAS mutant transgenic mice.The table displays KRAS mutation information, CP110 scoring and intensity of CP110 immunostaining in 6 lung cancer specimens from cyclin-E transgenic mice and Kras LSL-G12D driven engineered mice respectively.

Supplemental Fig. S4. (A) Effect of engineered KRAS expression on response to CDK2 inhibition by seliciclib. KRAS mutation sensitized lung cancer cells towards seliciclib-mediated CDK2 inhibition of growth as compared to control-ED-1 cells. (B) Cells transfected with the plasmid expressing HA-tagged human CP110 were used for detection of CP110 using an anti-CP110 antibody (1:1000) and an anti-HA antibody (1:1000), respectively. The upper band detected by the anti-CP110 antibody specifically recognizes CP110. (C) Respective CP110 and RAS protein expression profile in murine lung cancer cell lines is shown. (D) Effect of KRAS knockdown at 48 hours (left panel) and 72 hours (right panel) on CP110 expression in the 344P cell line. (E) Effect of KRAS knockdown at 72 hours (left panel) and 96 hours (right panel) on CP110 expression in the Hop62 cell line.

Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite KRAS oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in in vivo murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of KRAS oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite KRAS oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.

Targeting epigenetic regulators, such as histone-modifying enzymes, provides novel strategies for cancer therapy. The GCN5 lysine acetyltransferase (KAT) functions together with MYC both during normal development and in oncogenesis. As transcription factors, MYC family members are difficult to target with small-molecule inhibitors, but the acetyltransferase domain and the bromodomain in GCN5 might provide alternative targets for disruption of MYC-driven functions. GCN5 is part of two distinct multiprotein histone-modifying complexes, SAGA and ATAC. This review summarizes key findings on the roles of SAGA and ATAC in embryo development and in cancer to better understand the functional relationships of these complexes with MYC family members, as well as their future potential as therapeutic targets.

Single-cell sequencing encompasses a variety of technologies that evaluate cells at the genomic, transcriptomic, epigenomic, and proteomic levels. Each of these levels can be split into additional techniques that enable specific and optimized sequencing for a specialized purpose. At the transcriptomic level, single-cell sequencing has been used to understand immune-malignant cell networks, as well as differences between primary versus metastatic tumors. At the genomic and epigenomic levels, single-cell sequencing technology has been used to study genetic mutations involved in tumor evolution or the reprogramming of regulatory elements present in metastasized disease, respectively. Lastly, at the proteomic level, single-cell sequencing has been used to identify biomarkers important for predicting patient prognosis, as well as biomarkers essential for evaluating optimal treatment strategies. Integrated databases and atlases, as a result of large sequencing experiments, provide a vast array of information that can be applied to various studies and accessed by researchers to further answer scientific questions. This review summarizes recent, high-impact literature covering these aspects, as well as single-cell sequencing in the translational setting. Specifically, we review the potential that single-cell sequencing has in the clinic and its implementation in current clinical studies.

Simple Summary Despite being a group of rare diseases of mesenchymal origin, soft tissue sarcomas are heterogenous and display varying clinical behavior, and depending on the subtype, intermediate- and high-grade sarcomas have significant metastatic potential, making it difficult to establish a standardized therapy. Our work, as well as studies by others, emphasizes the high potential of immunotherapy for the treatment of sarcoma. The aim of this study was to determine whether specific genomic alterations, as well as the expression of infiltrating cytotoxic and suppressive cell type markers identified by next-generation sequencing (NGS), warrant further consideration of immunotherapy agents for treating certain soft tissue sarcoma subtypes. Altogether, our data provide a better understanding of the immune composition of different sarcoma subtypes to better identify novel therapy targets. Abstract Soft tissue sarcomas, depending on the subtype and grade, frequently recur and become metastatic after localized treatment. There is now great interest in applying immunotherapy to sarcomas to immuno-profile the different subtypes and immune monitor for prognosis. Our group previously showed that key immunotherapy target genes are present in sarcomas. Here, we extend our findings by demonstrating that sarcomas with a relatively high mutational load are likely to be more sensitive to immunotherapy compared to sarcomas with a lower mutation load. We also show that sarcomas with a higher mutation load are associated with the expression of key immune-related genes. We found that CD8+ T cells are present in sarcoma subtypes and that PD-L2 is highly expressed. These findings further define potential mechanisms behind the immunotherapy response of specific sarcoma subtypes and can be used to develop more optimal treatments in the future.

The spindle assembly checkpoint maintains genomic integrity. A key component is tyrosine threonine kinase (TTK, also known as Mps1). TTK antagonism is hypothesized to cause genomic instability and cell death. Interrogating The Cancer Genome Atlas revealed high TTK expression in lung adenocarcinomas and squamous cell cancers versus the normal lung (P < 0.001). This correlated with an unfavorable prognosis in examined lung adenocarcinoma cases (P = 0.007). TTK expression profiles in lung tumors were independently assessed by RNA in situ hybridization. CFI-402257 is a highly selective TTK inhibitor. Its potent antineoplastic effects are reported here against a panel of well-characterized murine and human lung cancer cell lines. Significant antitumorigenic activity followed independent treatments of athymic mice bearing human lung cancer xenografts (6.5 mg/kg, P < 0.05; 8.5 mg/kg, P < 0.01) and immunocompetent mice with syngeneic lung cancers (P < 0.001). CFI-402257 antineoplastic mechanisms were explored. CFI-402257 triggered aneuploidy and apoptotic death of lung cancer cells without changing centrosome number. Reverse phase protein arrays (RPPA) of vehicle versus CFI-402257–treated lung cancers were examined using more than 300 critical growth-regulatory proteins. RPPA bioinformatic analyses discovered CFI-402257 enhanced MAPK signaling, implicating MAPK antagonism in augmenting TTK inhibitory effects. This was independently confirmed using genetic and pharmacologic repression of MAPK that promoted CFI-402257 anticancer actions. TTK antagonism exerted marked antineoplastic effects against lung cancers and MAPK inhibition cooperated. Future work should determine whether CFI-402257 treatment alone or with a MAPK inhibitor is active in the lung cancer clinic.

GCN5, the catalytic subunit in the acetyltransferase modules of SAGA and ATAC, functions as a coactivator of gene transcription. The SAGA complex is recruited to chromatin by transcription factors such as MYC and E2F1 to facilitate acetylation of histones, especially H3 at lysine 9 (H3K9). Burkitt lymphoma is an aggressive subtype of Non-Hodgkin lymphoma driven by the overexpression of MYC. Comparison of GCN5 expression in normal human B cells versus human Burkitt Lymphoma cell lines indicates overexpression of GCN5 in lymphoma. Treatment of Burkitt lymphoma cell lines with a specific inhibitor indicates that decreased GCN5 HAT activity reduces viability and proliferation of these cells. Inhibition of GCN5 HAT activity also induces apoptosis in lymphoma cells. Expression of MYC target genes as well as genes associated with B cell receptor signaling are significantly downregulated upon inhibition of GCN5 enzymatic activity. This downregulation leads to diminished PI3K signaling, a critical pathway in lymphomagenesis. Our data indicate that inhibition of GCN5 HAT activity reduces the tumorigenic properties of human Burkitt lymphoma cells by attenuating BCR signaling and that GCN5 may be a viable target for lymphoma drug therapy.

Lung cancers contribute to the greatest number of cancer-related deaths worldwide and still pose challenges in response to current treatment strategies. Non-small cell lung cancer (NSCLC) accounts for over 85% of lung cancers diagnosed in the United States and novel therapeutics are needed for the treatment of this disease. First and second generation targeted therapies against specific mutated or rearranged oncogenes in NSCLCs show anti-tumor activity and also increase survival. However, many NSCLC patients eventually develop resistance to these therapies or do not properly respond if they have central nervous system metastases. Thus, this review summarizes recent developments and findings related to the generation of novel targeted therapies recently or currently being developed to tackle hurdles that prior therapies were not able to overcome.

Ubiquitin specific peptidase 18 (USP18), previously known as UBP43, is the IFN-stimulated gene 15 (ISG15) deconjugase. USP18 removes ISG15 from substrate proteins. This study reports that USP18-null mice (vs. wild-type mice) exhibited lower lipolysis rates, altered fat to body weight ratios, and cold sensitivity. USP18 is a regulator of lipid and fatty acid metabolism. Prior work established that USP18 promotes lung tumorigenesis. We sought to learn whether this occurs through altered lipid and fatty acid metabolism. Loss of USP18 repressed adipose triglyceride lipase (ATGL) expression; gain of USP18 expression upregulated ATGL in lung cancer cells. The E1-like ubiquitin activating enzyme promoted ISG15 conjugation of ATGL and destabilization. Immunoprecipitation assays confirmed that ISG15 covalently conjugates to ATGL. Protein expression of thermogenic regulators was examined in brown fat of USP18-null versus wild-type mice. Uncoupling protein 1 (UCP1) was repressed in USP18-null fat. Gain of USP18 expression augmented UCP1 protein via reduced ubiquitination. Gain of UCP1 expression in lung cancer cell lines enhanced cellular proliferation. UCP1 knockdown inhibited proliferation. Beta-hydroxybutyrate colorimetric assays performed after gain of UCP1 expression revealed increased cellular fatty acid beta-oxidation, augmenting fatty acid beta-oxidation in Seahorse assays. Combined USP18, ATGL, and UCP1 profiles were interrogated in The Cancer Genome Atlas. Intriguingly, lung cancers with increased USP18, ATGL, and UCP1 expression had an unfavorable survival. These findings reveal that USP18 is a pharmacologic target that controls fatty acid metabolism. Implications: USP18 is an antineoplastic target that affects lung cancer fatty acid metabolism.

Simple Summary Increased levels of the Usp22 deubiquitinase have been observed in several types of human cancer, particularly highly aggressive, therapy-resistant tumors. However, the role of Usp22 overexpression in cancer etiology is not known. To address whether Usp22 overexpression is sufficient to induce tumors in vivo, we created a mouse that expresses high levels of Usp22 in all tissues beginning in embryogenesis through to adulthood. Analyses of these mice clearly show that while Usp22 overexpression alters several tumor-related signaling pathways and induces associated morphological changes such as hyperbranching of the mammary gland, Usp22 overexpression alone is not sufficient to induce tumorigenesis. These findings indicate that Usp22 likely enhances tumor formation and progression through cooperation with oncoproteins that exacerbate abnormal signal transduction. Abstract Usp22 overexpression is observed in several human cancers and is correlated with poor patient outcomes. The molecular basis underlying this correlation is not clear. Usp22 is the catalytic subunit of the deubiquitylation module in the SAGA histone-modifying complex, which regulates gene transcription. Our previous work demonstrated that the loss of Usp22 in mice leads to decreased expression of several components of receptor tyrosine kinase and TGFβ signaling pathways. To determine whether these pathways are upregulated when Usp22 is overexpressed, we created a mouse model that expresses high levels of Usp22 in all tissues. Phenotypic characterization of these mice revealed over-branching of the mammary glands in females. Transcriptomic analyses indicate the upregulation of key pathways involved in mammary gland branching in mammary epithelial cells derived from the Usp22-overexpressing mice, including estrogen receptor, ERK/MAPK, and TGFβ signaling. However, Usp22 overexpression did not lead to increased tumorigenesis in any tissue. Our findings indicate that elevated levels of Usp22 are not sufficient to induce tumors, but it may enhance signaling abnormalities associated with oncogenesis.

KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the IFN-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having antineoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biologic consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo. To solidify this association, loss of Usp18 in KrasLA2/+/Usp18−/− mice was found to significantly reduce lung cancers as compared with parental KrasLA2/+ mice. Finally, translational relevance was confirmed in a human lung cancer panel by showing that USP18 IHC expression was significantly higher in KRAS-mutant versus wild-type lung adenocarcinomas. Implications: Taken together, this study highlights a new way to combat the oncogenic consequences of activated KRAS in lung cancer by inhibiting the DUB USP18. Mol Cancer Res; 15(7); 905–14. ©2017 AACR.

Simple Summary Since the Kirsten rat sarcoma viral oncogene homolog (KRAS) is mutated in about 25% of all human cancers and is at the center of pathways involved in tumorigenesis, it is necessary to compile and highlight the novel therapeutic strategies behind targeting this oncoprotein in cancer. Over the years, many have studied various methods to directly target KRAS with no success. Fortunately, there has been more success in targeting other proteins along the RAS pathway to yield a therapeutic response. However, some recent findings show promising results indicating that we are one step closer to developing an effective inhibitor that directly targets KRAS. The review presented here summarizes these recent findings and emphasizes the need to continue the search for the most optimal KRAS inhibitor that can be used to treat and potentially even cure certain tumor types. Abstract The Kirsten rat sarcoma viral oncogene homolog (KRAS) is mutated in approximately 25% of all human cancers and is known to be a major player promoting and maintaining tumorigenesis through the RAS/MAPK pathway. Over the years, a large number of studies have identified strategies at different regulatory levels to tackle this ‘difficult-to-target’ oncoprotein. Yet, the most ideal strategy to overcome KRAS and its downstream effects has yet to be uncovered. This review summarizes the role of KRAS activating mutations in multiple cancer types as well as the key findings for potential strategies inhibiting its oncogenic behavior. A comprehensive analysis of the different pathways and mechanisms associated with KRAS activity in tumors will ultimately pave the way for promising future work that will identify optimum therapeutic strategies.

The ubiquitin-like modifier interferon-stimulated gene 15 (ISG15) is implicated in both oncogenic and tumor suppressive programs. Yet, few ISGylation substrates are known and functionally validated in cancer biology. We previously found specific oncoproteins were substrates of ISGylation and were stabilized by the ISG15-specific deubiquitinase (DUB) ubiquitin specific peptidase 18 (USP18). Using reverse-phase protein arrays (RPPAs), this study reports that engineered loss of the DUB USP18 destabilized the tumor suppressor protein phosphatase and tensin homologue (PTEN) in both murine and human lung cancer cell lines. In contrast, engineered gain of USP18 expression in these same lung cancer cell lines stabilized PTEN protein. Using the protein synthesis inhibitor cycloheximide (CHX), USP18 knockdown was shown to destabilize PTEN whereas USP18 overexpression stabilized PTEN protein. Interestingly, repression of USP18 decreased cytoplasmic PTEN relative to nuclear PTEN protein levels. We sought to identify mechanisms engaged in this PTEN protein destabilization using immunoprecipitation assays and found ISG15 directly conjugated with PTEN. To confirm translational relevance of this work, USP18 and PTEN immunohistochemical expression were compared in comprehensive lung cancer arrays. There was a significant (P < 0.0001) positive correlation and association between PTEN and USP18 protein expression profiles in human lung cancers. Taken together, this study identified PTEN as a previously unrecognized substrate of the ISGylation post-translational modification pathway. The deconjugase USP18 serves as a novel regulator of PTEN stability. This indicates inhibition of ISGylation is therapeutically relevant in cancers.

Despite advances in targeted therapy, lung cancer remains the most common cause of cancer-related mortality in the United States. Chromosomal instability is a prominent feature in lung cancer and, because it rarely occurs in normal cells, it represents a potential therapeutic target. Our prior work discovered that lung cancer cells undergo anaphase catastrophe in response to inhibition of cyclin-dependent kinase 2 (CDK2), followed by apoptosis and reduced growth. In this study, the effects and mechanisms of the multi-CDK inhibitor dinaciclib on lung cancer cells were investigated. We sought to determine the specificity of CDK-dependent induction of anaphase catastrophe. Live cell imaging provided direct evidence that dinaciclib caused multipolar cell divisions resulting in extensive chromosome missegregation. Genetic knockdown of dinaciclib CDK targets revealed that repression of CDK2 and CDK1, but not CDK5 or CDK9, triggered anaphase catastrophe in lung cancer cells. Overexpression of CP110, which is a mediator of CDK2 inhibitor–induced anaphase catastrophe (and a CDK1 and 2 phosphorylation substrate), antagonized anaphase catastrophe and apoptosis following dinaciclib treatment. Consistent with our previous findings, acquisition of activated KRAS sensitized lung cancer cells to dinaciclib-mediated anaphase catastrophe and cell death. Combining dinaciclib with the mitotic inhibitor taxol augmented anaphase catastrophe induction and reduced cell viability of lung cancer cells. Thus, the multi-CDK inhibitor dinaciclib causes anaphase catastrophe in lung cancer cells and should be investigated as a potential therapeutic for wild-type and KRAS-mutant lung cancer, individually or in combination with taxanes. Mol Cancer Ther; 15(11); 2758–66. ©2016 AACR.

This Special Issue on “Single-cell Data Science” aims to highlight recent advances in the area of single-cell sequencing technologies and data analytics [...]

Ubiquitin specific peptidase 18 (USP18) is the Interferon-Stimulated Gene 15 (ISG15) deconjugase that stabilizes target proteins by removing ISG15 from substrate proteins. We found that USP18 null mice have lower lipolysis rates, altered fat to body weight ratios and marked cold sensitivity. These findings implicated USP18 as a regulator of lipid and fatty acid metabolism. Prior work established that USP18 can promote lung tumorigenesis. We examined whether this occurs in lung cancer cells through altered lipid and fatty acid metabolism. The siRNA knockdown of USP18 downregulated Adipose Triglyceride Lipase (ATGL) and this reduced lipolysis. In contrast, engineered gain of USP18 expression upregulated ATGL and increased lipolysis. Notably, Ubiquitin Activating Enzyme E1 Like Protein (UBE1L) promoted ISG15-conjugated destabilization of ATGL protein. Immunoprecipitation experiments confirmed that ISG15 can covalently conjugate to ATGL. Protein expression profiles of thermogenic regulators were examined in brown fat of USP18 null versus wild-type mice. Strikingly, the master thermoregulatory protein Uncoupling Protein 1 (UCP1) was markedly repressed in brown fat of USP18 null mice. In contrast, USP18 reconstitution augmented UCP1 levels. Engineered gain of USP18 expression stabilized UCP1 protein through reduced Ubiquitin conjugation to UCP1 in the examined lung cancer cell lines. Intriguingly, gain of UCP1 expression in both human and murine lung cancer cell lines promoted cellular proliferation and UCP1 knockdown reduced proliferation. The beta-hydroxybutyrate colorimetric assay determined that UCP1 overexpression promoted fatty acid beta-oxidation in lung cancer cells. Seahorse assays independently confirmed that gain of UCP1 expression increased fatty acid beta-oxidation. Expression profiles of USP18, ATGL and UCP1 species were interrogated using The Cancer Genome Atlas (TCGA). Lung adenocarcinoma and squamous cell carcinoma cases having higher USP18, ATGL and UCP1 levels had a statistically-significantly unfavorable survival. This association also occurred in renal chromophobe carcinoma and low grade gliomas. Together, these findings implicate the deubiquitinase USP18 as a novel molecular pharmacologic target that controls fatty acid metabolism as an energy source for lung cancer growth. These observations provide a strong rationale to uncover USP18 pharmacologic inhibitors that would combat lung and potentially other human cancers. Citation Format: Xi Liu, Yun Lu, Zibo Chen, Xiuxia Liu, Weiguo Hu, Lin Zheng, Yulong Chen, Mi Shi, Lisa Mustachio, Jason Roszik, Masanori Kawakami, Sarah Freemantle, Ethan Dmitrovsky. USP18 promotes lipolysis, fatty acid oxidation and lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2547.

In “CFI-400945 is not a selective cellular PLK4 inhibitor,” Oegema et al. (1) raise thoughtful comments about our article (2). We appreciate their interest and critique. They propose that CFI-400945 activity was not from Polo-like kinase (PLK4) inhibition and argue that antineoplastic activity was through Aurora B kinase (1), rather than PLK4 inhibition (2). Kinases often share related catalytic pockets, and targeting one kinase without affecting another is difficult to achieve. This is true for CFI-400945 (2) and centrinone, the PLK4 inhibitor (3) used by Oegema et al. (1). CFI-400945 is (by IC50) 38-fold more potent in causing PLK4 inhibition than in antagonizing Aurora B kinase (4, 5). Despite their claim … [↵][1]1Email: tak.mak{at}uhnresearch.ca. [1]: #xref-corresp-1-1

Cancer cells often have supernumerary centrosomes that promote genomic instability, a pathognomonic feature of cancer. During mitosis, cancer cells with supernumerary centrosomes undergo bipolar cell division by clustering centrosomes into two poles. When supernumerary centrosome clustering is antagonized, cancer cells are forced to undergo multipolar division leading to death of daughter cells. This proapoptotic pathway, called anaphase catastrophe, preferentially eliminates aneuploid cancer cells and malignant tumors in engineered mouse models. Anaphase catastrophe occurs through the loss or inhibition of the centrosomal protein CP110, a direct cyclin-dependent kinase 1 (CDK1) and CDK2 target. Intriguingly, CP110 is repressed by the KRAS oncoprotein. This sensitizes KRAS-driven lung cancers (an unmet medical need) to respond to CDK2 inhibitors. Anaphase catastrophe-inducing agents like CDK1 and CDK2 antagonists are lethal to cancer cells with supernumerary centrosomes, but can relatively spare normal cells with two centrosomes. This mechanism is proposed to provide a therapeutic window in the cancer clinic following treatment with a CDK1 or CDK2 inhibitor. Taken together, anaphase catastrophe is a clinically tractable mechanism that promotes death of neoplastic tumors with aneuploidy, a hallmark of cancer. Mol Cancer Ther; 17(4); 724–31. ©2018 AACR.

Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Prior work was substantially extended by showing CFI-400945 causes polyploidy, growth inhibition, and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with >4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in lung cancer cells. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was significantly inhibited at well-tolerated dosages. Phosphohistone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05). Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism cooperated with PLK4 inhibition. Taken together, PLK4 inhibition alone or as part of a combination regimen is a promising way to combat lung cancer.

Chromosomal instability (CIN) is a hallmark of solid tumor biology and is implicated in carcinogenesis. Preferentially eliminating malignant cells by targeting CIN and aneuploidy is an attractive antineoplastic strategy. We previously reported that CDK2 antagonism causes lung cancer cells to undergo anaphase catastrophe and apoptosis through inhibition of phosphorylation of the centrosomal protein CP110. Cells with activating KRAS mutations were particularly sensitive to CDK2 inhibition due to downregulation of CP110 protein levels. This study investigated mechanisms of CDK2 antagonism that mediate anaphase catastrophe via changes in CP110 protein expression and how activated KRAS affects CP110 levels in lung cancers. Site-directed mutagenesis revealed candidate CDK phosphorylation sites of CP110 (residues Ser 170 and Thr 194) critical for conferring anaphase catastrophe by altering centrosome clustering in mitosis. Intriguingly, KRAS mutation can promote CP110 protein degradation by upregulating the ubiquitin ligase SCFcyclinF, which targets CP110 protein for destabilization. Finally, CDK2 inhibitor response was enhanced when combined with knockdown of the deubiquitinase USP33 that in turn accelerates CP110 protein degradation. Thus, this study provides molecular pharmacologic insights into how CP110 expression regulates response to CDK2 inhibition. An improved understanding of in vitro antineoplastic mechanisms of combining CDK2 antagonism with induced CP110 repression provides a rationale for exploring clinical consequences of this strategy. Taken together, preclinical findings obtained from combining CDK2 inhibition with USP33 repression have implications for treating patients with non–small cell lung cancers. Mol Cancer Ther; 14(11); 2576–85. ©2015 AACR.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 2029–38. ©2015 AACR.

Ubiquitination and ubiquitin-like posttranslational modifications (PTM) regulate activity and stability of oncoproteins and tumor suppressors. This implicates PTMs as antineoplastic targets. One way to alter PTMs is to inhibit activity of deubiquitinases (DUB) that remove ubiquitin or ubiquitin-like proteins from substrate proteins. Roles of DUBs in carcinogenesis have been intensively studied, yet few inhibitors exist. Prior work provides a basis for the ubiquitin-specific protease 18 (USP18) as an antineoplastic target. USP18 is the major DUB that removes IFN-stimulated gene 15 (ISG15) from conjugated proteins. Prior work discovered that engineered loss of USP18 increases ISGylation and in contrast to its gain decreases cancer growth by destabilizing growth-regulatory proteins. Loss of USP18 reduced cancer cell growth by triggering apoptosis. Genetic loss of USP18 repressed cancer formation in engineered murine lung cancer models. The translational relevance of USP18 was confirmed by finding its expression was deregulated in malignant versus normal tissues. Notably, the recent elucidation of the USP18 crystal structure offers a framework for developing an inhibitor to this DUB. This review summarizes strong evidence for USP18 as a previously unrecognized pharmacologic target in oncology. Cancer Res; 78(3); 587–92. ©2018 AACR.

Metastasis is a major cause of cancer-related mortality. Yet, current antineoplastic strategies do not effectively repress metastasis. Thus, there is a pressing need to explore targets that can reduce metastasis, especially in lung cancer that is the most common cause of cancer mortality. Our prior work reported that expression of the ubiquitin-specific peptidase 18 (USP18) was augmented in diverse cancers (including lung cancers) and USP18 knock-down suppressed in vitro and in vivo growth of murine and human lung cancers. This deubiquitinase stabilizes target proteins by removing interferon-stimulated gene 15 (ISG15) from substrate proteins. We explored here whether engineered loss of USP18 decreased lung cancer metastasis. Stable USP18 knockdown was independently achieved in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells by use of small hairpin RNAs (shRNAs). Notably, knockdown of USP18 expression reduced lung cancer cellular proliferation, migration and invasion versus control shRNA transfectants (p < 0.001). Lung cancer cells selected for high metastatic potential (344SQ and KC2) were independently injected via tail vein or subcutaneously to establish metastatic mouse models. USP18 knockdown statistically-significantly decreased lung cancer metastases as compared to control shRNA transfectants. To elucidate engaged mechanisms, Reverse Phase Protein Array (RPPA) analysis was performed to interrogate over 300 growth-regulatory proteins in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells having engineered USP18 knockdown. Results were compared to control shRNA transfectants. Ingenuity pathway analysis highlighted fatty acid synthase (FASN) as a potential USP18 target that regulated lung cancer metastasis. Independent immunoblot analysis confirmed FASN expression was reduced in cancers once USP18 knockdown was achieved. Functional validation of FASN in lung metastasis is underway. These and other findings that will be presented provide a strong rationale for targeting the deubiquitinase USP18 to reduce lung cancer metastasis and combat lung cancers. Citation Format: Zibo Chen, Lin Zheng, Yulong Chen, Masanori Kawakami, Lisa Mustachio, Jason Roszik, Xin Liu, Jonathan Kurie, Xi Liu, Ethan Dmitrovsky. Loss of the ubiquitin-specific peptidase 18 (USP18) reduces lung cancer metastasis [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 170.

Genetically-unstable cancer cells have supernumerary centrosomes and aneuploidy (a hallmark of cancer). CDK2 antagonism inhibits clustering of excessive centrosomes at mitosis leading to multipolar cell division and apoptotic death. This process is called anaphase catastrophe. Anaphase catastrophe affects preferentially aneuploid cancer cells while sparing normal cells with two centrosomes. CYC065 (Cyclacel) is a next-generation CDK2/9 inhibitor that is undergoing clinical trials. Here, we explored CYC065 activity against aneuploid lung and other cancers. CYC065 substantially inhibited growth, triggered apoptosis, and induced anaphase catastrophe in murine (ED1, LKR13, and 393P) and human (Hop62, A549, and H1299) lung cancer cells. In marked contrast, these effects were largely unseen in bipolar immortalized pulmonary epithelial (murine C10 and human BEAS-2B) cells. Notably, anaphase catastrophe was induced by CYC065-treatment even in pancreatic and colon cancers that are often driven by the KRAS oncoprotein. This suggests that engaging anaphase catastrophe is a general antineoplastic mechanism. Comprehensive expression analysis of nearly 300 growth-regulatory proteins was performed after CYC065 treatment in lung cancer cells using Reverse Phase Protein Arrays (RPPAs). In addition to known CDK targets (such as phosphorylated retinoblastoma protein), FAK phosphorylation and SAC phosphorylation that are known to regulate metastasis were unexpectedly down-regulated. Consistent with this finding, in vitro migration and invasion CYC065 treatment assays markedly inhibited migration and invasion of lung cancer cells. CYC065 anti-neoplastic effects were interrogated in mice. CYC065 significantly inhibited tumor growth in several lung cancer syngeneic xenografts and patient-derived xenografts (PDXs) and reduced metastasis of 344SQ lung cancer cells in a syngeneic tail-vein injection model. Immunohistochemistry (IHC) studies of resected PDX tumors revealed downregulation of p-CDK2 (Thr160) and MCL1 expression after CYC065 treatment which is consistent with CDK2/9 antagonism, respectively. In summary, CYC065 engages anaphase catastrophe and elicits marked antineoplastic effects in diverse cancers. These effects are due to mechanisms that confer after CDK2/9 inhibition, anaphase catastrophe, MCL1 down-regulation and suppression of proteins that regulate metastasis. Taken together, findings reveal that anaphase catastrophe is a general antineoplastic mechanism engaged in aneuploid cancers. Intriguingly, this mechanism is activated after CDK2/9 inhibition of lung and other cancers, including those driven by the KRAS oncoprotein. Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Yulong Chen, Zibo Chen, Jason Roszik, Xi Liu, Ethan Dmitrovsky. Next generation CDK2/9 inhibitor CYC065 triggers anaphase catastrophe in diverse aneuploid cancers and markedly inhibits growth and metastasis [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 4407.

Lung cancer causes the highest mortality in cancer-related deaths. Definition of novel molecular targets is needed since these cancers often become resistant to existing therapies. Epigenetic modifications may provide such targets. Recent reports suggest that the histone acetyltransferase (HAT) module within the transcriptional coactivator SAGA complex plays a role in cancer, creating a new link between epigenetic regulators and this disease. We aim to define the role of the SAGA HAT protein GCN5 in the regulation and function of oncogenes in lung cancers. Since prior work indicates that GCN5 regulates the stability of c-MYC and serves as a coactivator for MYC target genes, we hypothesize that there is a positive relationship between GCN5 and c-MYC in non-small cell lung cancer (NSCLC). We also propose that inhibition of GCN5 will reduce lung cancer formation and progression in mouse models of lung cancer driven by c-MYC overexpression. Our data indicate that both GCN5 and c-MYC proteins are upregulated in NSCLC cells compared to normal lung epithelial cell lines. This trend is observable only at the protein level, indicating that the upregulation of these proteins in NSCLC cells occurs post-transcriptionally. Both GCN5 and c-MYC expression levels are also positively associated in mouse and human NSCLC cell lines. Genetic repression of GCN5 in NSCLC cells reduces cell proliferation, increases the population of necrotic cells, and reduces c-MYC expression. Inhibition of the GCN5 active site using commercially available probes in NSCLC cells significantly (p < 0.05) reduces cell proliferation, increases the percentage of cells undergoing apoptosis, and reduces c-MYC expression. In human NSCLC tissues, GCN5 and c-MYC positively correlate and are associated with MYC target genes. Our ongoing studies will determine whether targeting GCN5 represses multiple oncogenic pathways in cancer and whether it inhibits oncoproteins difficult to directly target, such as MYC. We expect that our findings will highlight the importance of epigenetic regulators in carcinogenesis and extend beyond our current research focus to other cancer types. Citation Format: Lisa Maria Mustachio, Jason Roszik, Aimee Farria, Sharon YR Dent. GCN5 positively correlates with c-MYC in non-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 4333.

Background The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel). Methods CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided. Results CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 μM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 μM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 μM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 μM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 μM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065. Conclusions Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.

Vibrio cholerae continues to pose a health threat in many developing nations and regions of the world struck by natural disasters. It is a pathogen that rapidly adapts to aquatic environments and the human small intestine. Small regulatory RNAs (sRNAs) may contribute to this adaptability. Specifically, the mannitol operon sRNA (MtlS sRNA; previously designated the IGR7 sRNA) is transcribed antisense to the 5′ untranslated region of the mtl operon, encoding the mannitol-specific phosphotransferase system. Mannitol is a six-carbon sugar alcohol that accumulates in the human small intestine, the primary site of V. cholerae colonization. To better understand the V. cholerae mtl operon at a molecular level, we investigated mtlA expression in the presence of various carbon sources and the role of the MtlS sRNA. We observed that MtlA protein is present only in cells grown on mannitol sugar, whereas MtlS sRNA is expressed during growth on all sugars other than mannitol. In contrast, mtlA mRNA is expressed in similar amounts regardless of the carbon source used for bacterial growth. These observations suggest that the regulation of MtlA protein expression is a posttranscriptional event. We further demonstrate that MtlS sRNA overexpression repressed MtlA synthesis without affecting the stability of the messenger and that this process is largely independent of Hfq. We propose a model in which, when carbon sources other than mannitol are present, MtlS sRNA is transcribed, base pairs with the 5′ untranslated region of the mtlA mRNA, occluding the ribosome binding site, and inhibits the synthesis of the mannitol-specific phosphotransferase system.

USP18 is the ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We found USP18 null mice are markedly cold sensitive versus their wild-type littermates. USP18 null mice had significantly (P < 0.05) greater temperature declines than wild-type mice. These mice also had lower lipolysis rates and altered fat to body weight ratios. This implicated USP18 as a regulator of lipid and fatty acid metabolism. Protein expression profiles of thermogenic regulators were examined in brown fat of USP18 null versus wild-type mice. Strikingly, the thermoregulatory protein UCP-1 was substantially repressed in brown fat of USP18 null mice. To establish that UCP-1 repression was caused by loss of USP18, stable USP18 knock-down was independently achieved in a panel of murine lung cancer cell lines using transfected small hairpin RNAs (shRNAs). USP18 down-regulation by different shRNAs reduced UCP-1 levels as compared to controls. Engineered gain of USP18 expression stabilized UCP-1 expression in these lung cancer cell lines. UCP-1 destabilization followed complex formation with the ubiquitin-like protein ISG15. Immunoprecipitation assays established that complexes formed between ISG15 and UCP-1. Prior work found that USP18 regulates lung tumorigenesis. We therefore examined UCP-1 levels in human lung adenocarcinomas using The Cancer Genome Atlas (TCGA). Lung adenocarcinomas with undetected UCP-1 expression had significantly improved survival versus cases that expressed UCP-1. Notably, gain of UCP-1 expression in both human and murine lung cancer cell lines promoted their growth. In marked contrast, UCP-1 knockdown reduced proliferation. We explored whether UCP-1 expression affected fatty acid metabolism. The beta-hydroxybutyrate (ketone body) colorimetric assay confirmed that UCP-1 overexpression promoted fatty acid beta-oxidation. Gain of UCP-1 expression also increased fatty acid beta-oxidation using seahorse assays. Thus, a direct link exists between USP18 and expression of the thermoregulator UCP-1. This affects UCP-1 stability and lung cancer growth by altering fatty acid metabolism. Together, these findings implicate the deubiquitinase USP18 as a novel molecular pharmacologic target that controls fatty acid metabolism as an energy source for lung cancer growth. Citation Format: Xi Liu, Yun Lu, Weiguo Hu, Zibo Chen, Lisa M. Mustachio, Jason Roszik, Lin Zheng, Masanori Kawakami, Yulong Chen, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of ubiquitin-specific peptidase 18 (USP18) destabilizes the regulator of thermogenesis uncoupling protein-1 (UCP-1) and represses lung cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2399.

Lung cancer causes the highest mortality in cancer-related deaths. Definition of novel molecular targets is needed since these cancers often become resistant to existing therapies. Epigenetic modifications may provide such targets. Recent reports suggest that the histone acetyltransferase (HAT) module within the transcriptional coactivator SAGA complex plays a role in cancer, creating a new link between epigenetic regulators and this disease. We are working to define the role of the SAGA HAT protein GCN5 in the regulation and function of oncogenes in lung cancers. Since our prior work indicates that GCN5 serves as a coactivator for MYC target genes, we hypothesize that inhibition of GCN5 will reduce lung oncogenesis by suppressing MYC functions. We also propose that inhibition of GCN5 will reduce lung cancer formation and progression in mouse models of lung cancer. Our preliminary data indicate that both GCN5 and MYC expression levels are upregulated and positively associated in mouse and human lung cancers. Both proteins are also associated with MYC target genes in lung cancer tissues. Inhibition of the GCN5 active site or bromodomain significantly (p < 0.05) reduces cell proliferation compared to normal lung epithelium cells. Studies investigating how GCN5 inhibition affects other lung cancer phenotypes are underway. Specific genes and pathways affected by GCN5 loss will be determined using RNA-Seq and RPPA assays to identify mechanisms behind observed phenotypes. In addition, we will determine whether knockout of Gcn5 reduces lung formation and progression in the KrasLA2 lung cancer mouse model. Our ongoing studies will determine whether targeting GCN5 represses multiple oncogenic pathways in cancer and whether it inhibits oncoproteins difficult to directly target, such as MYC. We expect that our findings will highlight the importance of epigenetic regulators in carcinogenesis and extend beyond our current research focus to other cancer types. Citation Format: Lisa Maria Mustachio, Jason Roszik, Sharon YR Dent. Role of the SAGA histone acetylation module in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 363.

Despite current treatments, lung cancers remain a major public health problem. Innovative ways are needed to treat or prevent these cancers. Centrosomes are critical for fidelity of mitosis. Abnormal centrosome numbers can cause aberrant mitosis and cell death. Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication and its deregulation alters centrosome number and mitosis. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Here, we explored CFI-400945 activity against lung cancer using in vitro and in vivo models. CFI-400945 caused polyploidy, growth inhibition and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with > 4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored by individually staining treated and control cells with γ-tubulin, α-tubulin, and DAPI. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in the examined lung cancer cell lines. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was statistically significantly reduced at dosages that were well tolerated. Phospho-histone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05) indicating the clinical relevance of PLK4 expression in lung cancer. Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism (that confers multipolar anaphase catastrophe by inhibiting centrosome clustering) showed synergistic effects (CI < 1) on lung cancer cell growth when combined with CDI-400945. This is an appealing cooperating regimen since each agent increases multipolar cell division and cancer cell death. CFI-400945 is undergoing phase I clinical trial testing (NCT01954316). Taken together, targeting PLK4 for inhibition holds promise for lung cancer therapy either as a single agent or when combined with another agent that deregulates mitosis. Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguez-Canales, Barbara Mino, Jonathan M. Kurie, Jason Roszik, Pamela Andrea Villalobos, Kelsie L. Thu, David W. Cescon, Jennifer Silvester, Ignacio Wistuba, Tak W. Mak, Xi Liu, Ethan Dmitrovsky. Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1948.