Fadzai Chinyengetere

Blockade of the Ubiquitin Protease UBP43 Destabilizes Transcription Factor PML/RARα and Inhibits the Growth of Acute Promyelocytic Leukemia

More effective treatments for acute promyelocytic leukemia (APL) are needed. APL cell treatment with all-trans-retinoic acid (RA) degrades the chimeric, dominant-negative-acting transcription factor promyelocytic leukemia gene (PML)/RARα, which is generated in APL by chromosomal translocation. The E1-like ubiquitin-activating enzyme (UBE1L) associates with interferon-stimulated gene ISG15 that binds and represses PML/RARα protein. Ubiquitin protease UBP43/USP18 removes ISG15 from conjugated proteins. In this study, we explored how RA regulates UBP43 expression and the effects of UBP43 on PML/RARα stability and APL growth, apoptosis, or differentiation. RA treatment induced UBE1L, ISG15, and UBP43 expression in RA-sensitive but not RA-resistant APL cells. Similar in vivo findings were obtained in a transgenic mouse model of transplantable APL, and in the RA response of leukemic cells harvested directly from APL patients. UBP43 knockdown repressed PML/RARα protein levels and inhibited RA-sensitive or RA-resistant cell growth by destabilizing the PML domain of PML/RARα. This inhibitory effect promoted apoptosis but did not affect the RA differentiation response in these APL cells. In contrast, elevation of UBP43 expression stabilized PML/RARα protein and inhibited apoptosis. Taken together, our findings define the ubiquitin protease UBP43 as a novel candidate drug target for APL treatment.

Evidence for the Ubiquitin Protease UBP43 as an Antineoplastic Target

New pharmacologic targets are needed for lung cancer. One candidate pathway to target is composed of the E1-like ubiquitin-activating enzyme (UBE1L) that associates with interferon-stimulated gene 15 (ISG15), which complexes with and destabilizes cyclin D1. Ubiquitin protease 43 (UBP43/USP18) removes ISG15 from conjugated proteins. This study reports that gain of UBP43 stabilized cyclin D1, but not other D-type cyclins or cyclin E. This depended on UBP43 enzymatic activity; an enzymatically inactive UBP43 did not affect cyclin D1 stability. As expected, small interfering RNAs that reduced UBP43 expression also decreased cyclin D1 levels and increased apoptosis in a panel of lung cancer cell lines. Forced cyclin D1 expression rescued UBP43 apoptotic effects, which highlighted the importance of cyclin D1 in conferring this. Short hairpin RNA-mediated reduction of UBP43 significantly increased apoptosis and reduced murine lung cancer growth in vitro and in vivo after transplantation of these cells into syngeneic mice. These cells also exhibited increased response to all-trans-retinoic acid, interferon, or cisplatin treatments. Notably, gain of UBP43 expression antagonized these effects. Normal-malignant human lung tissue arrays were examined independently for UBP43, cyclin D1, and cyclin E immunohistochemical expression. UBP43 was significantly (P < 0.01) increased in the malignant versus normal lung. A direct relationship was found between UBP43 and cyclin D1 (but not cyclin E) expression. Differential UBP43 expression was independently detected in a normal-malignant tissue array with diverse human cancers. Taken together, these findings uncovered UBP43 as a previously unrecognized antineoplastic target. Mol Cancer Ther; 11(9); 1968–77. ©2012 AACR.

Comparing Histone Deacetylase Inhibitor Responses in Genetically Engineered Mouse Lung Cancer Models and a Window of Opportunity Trial in Patients with Lung Cancer

Histone deacetylase inhibitor (HDACi; vorinostat) responses were studied in murine and human lung cancer cell lines and genetically engineered mouse lung cancer models. Findings were compared with a window of opportunity trial in aerodigestive tract cancers. In human (HOP62, H522, and H23) and murine transgenic (ED-1, ED-2, LKR-13, and 393P, driven, respectively, by cyclin E, degradation-resistant cyclin E, KRAS, or KRAS/p53) lung cancer cell lines, vorinostat reduced growth, cyclin D1, and cyclin E levels, but induced p27, histone acetylation, and apoptosis. Other biomarkers also changed. Findings from transgenic murine lung cancer models were integrated with those from a window of opportunity trial that measured vorinostat pharmacodynamic responses in pre- versus posttreatment tumor biopsies. Vorinostat repressed cyclin D1 and cyclin E expression in murine transgenic lung cancers and significantly reduced lung cancers in syngeneic mice. Vorinostat also reduced cyclin D1 and cyclin E expression, but increased p27 levels in post- versus pretreatment human lung cancer biopsies. Notably, necrotic and inflammatory responses appeared in posttreatment biopsies. These depended on intratumoral HDACi levels. Therefore, HDACi treatments of murine genetically engineered lung cancer models exert similar responses (growth inhibition and changes in gene expression) as observed in lung cancer cell lines. Moreover, enhanced pharmacodynamic responses occurred in the window of opportunity trial, providing additional markers of response that can be evaluated in subsequent HDACi trials. Thus, combining murine and human HDACi trials is a strategy to translate preclinical HDACi treatment outcomes into the clinic. This study uncovered clinically tractable mechanisms to engage in future HDACi trials. Mol Cancer Ther; 12(8); 1545–55. ©2013 AACR.

Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.

Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

BackgroundUSP18 (ubiquitin-specific protease 18) removes ubiquitin-like modifier interferon stimulated gene 15 (ISG15) from conjugated proteins. USP18 null mice in a FVB/N background develop tumors as early as 2 months of age. These tumors are leiomyosarcomas and thus represent a new murine model for this disease.MethodsHeterozygous USP18 +/− FVB/N mice were bred to generate wild-type, heterozygous and homozygous cohorts. Tumors were characterized immunohistochemically and two cell lines were derived from independent tumors. Cell lines were karyotyped and their responses to restoration of USP18 activity assessed. Drug testing and tumorigenic assays were also performed. USP18 immunohistochemical staining in a large series of human leiomyosacomas was examined.ResultsUSP18 −/− FVB/N mice spontaneously develop tumors predominantly on the back of the neck with most tumors evident between 6–12 months (80 % penetrance). Immunohistochemical characterization of the tumors confirmed they were leiomyosarcomas, which originate from smooth muscle. Restoration of USP18 activity in sarcoma-derived cell lines did not reduce anchorage dependent or independent growth or xenograft tumor formation demonstrating that these cells no longer require USP18 suppression for tumorigenesis. Karyotyping revealed that both tumor-derived cell lines were aneuploid with extra copies of chromosomes 3 and 15. Chromosome 15 contains the Myc locus and MYC is also amplified in human leiomyosarcomas. MYC protein levels were elevated in both murine leiomyosarcoma cell lines. Stabilized P53 protein was detected in a subset of these murine tumors, another feature of human leiomyosarcomas. Immunohistochemical analyses of USP18 in human leiomyosarcomas revealed a range of staining intensities with the highest USP18 expression in normal vascular smooth muscle. USP18 tissue array analysis of primary leiomyosarcomas from 89 patients with a clinical database revealed cases with reduced USP18 levels had a significantly decreased time to metastasis (P = 0.0441).ConclusionsUSP18 null mice develop leiomyosarcoma recapitulating key features of clinical leiomyosarcomas and patients with reduced-USP18 tumor levels have an unfavorable outcome. USP18 null mice and the derived cell lines represent clinically-relevant models of leiomyosarcoma and can provide insights into both leiomyosarcoma biology and therapy.

Deubiquitinase USP18 Loss Mislocalizes and Destabilizes KRAS in Lung Cancer

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.

Abstract 1615: Gene expression profile predicts response to smoothened inhibitors in epithelial cancers

The hedgehog (HH) signaling pathway is important for embryonic development as it controls cell fate and proliferation. Deregulation of the HH pathway is linked to the onset and maintenance of multiple cancers. Clinical trials revealed promising effects of targeting the HH pathway with smoothened (Smo) inhibitors, particularly in basal cell carcinomas and medulloblastomas where HH pathway activation occurs. This study sought to uncover a gene signature that would predict response to Smo inhibitors, such as cyclopamine or SANT-1. A panel of 705 human cancer cell lines from diverse epithelial cancers was interrogated for sensitivity to cyclopamine. A subset of cancer cell lines exhibited substantial growth inhibition across a broad array of epithelial cancers. Sensitivity was significantly linked to high Gli1, high cyclin E, and low insulin-like growth factor binding protein 6 (IGFBP6) expression levels (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1615. doi:10.1158/1538-7445.AM2011-1615

Abstract 1255: Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein

KRAS is frequently mutated in lung cancers. Innovative strategies are needed to combat KRAS mutant lung cancers because these tumors are often resistant to therapy. This study reports that loss of the deubiquitinase USP18 leads to destabilization of the KRAS oncogenic protein in panels of murine and human lung cancer cells. In marked contrast, engineered gain of USP18 expression using retroviral vectors introduced into the same panel of murine and human lung cancer cells stabilized KRAS protein. Loss of USP18 expression in KRAS mutant-expressing lung cancer cells inhibited their growth while gain of USP18 expression opposed this effect. We sought to identify mechanisms engaged in this KRAS protein destabilization. Intriguingly, immunoprecipitation assays established that KRAS conjugates with the ubiquitin-like protein ISG15. This leads to KRAS protein destabilization. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown was shown to destabilize mutant KRAS more prominently than wild-type KRAS protein. To independently confirm that KRAS directly complexes with ISG15, site-directed mutagenesis was performed to render the C-terminal domain of KRAS lysine-less. This led to stabilization of KRAS, despite knockdown of USP18. These studies were confirmed and extended in the in vivo setting of KRAS-driven lung cancers in KRASLA2/+ mice within the FVB strain background. We crossed these mice with USP18 null (USP18-/-) mice to obtain KRASLA2/+;USP18-/- compound mice. Strikingly, these compound mice had statistically significantly (P Citation Format: Lisa Maria Mustachio, Yun Lu, Laura J. Tafe, Angeline S. Andrew, Vincent Memoli, Jaime Rodriguez-Canales, Pamela A. Villalobos, Ignacio Wistuba, Jun Yu, Jack J. Lee, Fadzai Chinyengetere, David J. Sekula, Xi Liu, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1255.

Abstract 1782: The ubiquitin protease UBP43 is a target for KRAS mutant lung cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA KRAS is frequently mutated in human lung cancers. Lung cancers with mutant KRAS are often resistant to current therapies. There is therefore a need to develop new ways to target these tumors with KRAS mutations. We previously found that the protein modification pathway containing the ubiquitin-like interferon-stimulated gene 15 (ISG15) destabilizes cyclin D1 and represses growth and tumorigenicity of lung cancer cells. This study substantially extends that prior work by reporting that inhibition of the ubiquitin protease UBP43, which removes ISG15 from protein substrates, also destabilizes KRAS protein levels. In a screen of murine lung cancer cells with KRAS mutations, the lines with highest KRAS protein levels had the highest levels of UBP43 protein, implying a direct link between UBP43 and KRAS. Stable overexpression of UBP43 in murine lung cancer cells increased cancer cell growth and stability of KRAS protein. In contrast, knockdown of UBP43 in murine and human lung cancer cell lines had the opposite effects on growth and KRAS protein stability. Co-immunoprecipitation experiments established an interaction between ISG15 and both wild-type KRAS and mutant KRAS. UBP43 immunohistochemical staining of human lung cancer cases showed a marked increase in UBP43 expression in KRAS mutant versus KRAS wild-type lung cancers. Preliminary findings in newly engineered mouse models revealed an increase in lung cancers in KRAS mutant/UBP43 wild-type as compared to KRAS mutant/UBP43 null mice. This finding provides a genetic basis for targeting UBP43 in lung cancers. Taken together, these studies broaden the role of UBP43 as an antineoplastic target to include an unmet medical need. This is to combat lung cancers that harbor KRAS mutations. Citation Format: Lisa Maria Mustachio, Fadzai Chinyengetere, Yun Lu, Shanhu Hu, Masanori Kawakami, Laura J. Tafe, Alexey Danilov, David J. Sekula, Tian Ma, Sarah J. Freemantle, Ethan Dmitrovsky. The ubiquitin protease UBP43 is a target for KRAS mutant lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2014-1782

Abstract 1089: Spontaneous formation of sarcomas in Usp18 knockout mice.

Undifferentiated pleomorphic sarcoma (UPS) and leimyosarcoma are two of the most common adult soft tissue sarcomas. Their etiology is unknown hence the need for new models to elucidate the biology of these diseases. We report here a spontaneously occurring mouse model of sarcoma in Usp18 knockout mice. Usp18 is the deconjugase for isgylation. Isgylation is the ubiquitin-like protein modification in which ISG15 is conjugated to proteins. Usp18 removes ISG15 from the target proteins, thus reversing the functional consequences of ISG15 conjugation. Usp18 knockout mice on a C57Bl6 background developed neurological abnormalities and exhibited 100% mortality within 5 months. These mice were also hypersensitive to Type I IFN stimulation, which was attributed to an increased JAK-STAT signaling. We now report that Usp18 knockout mice in a FVB background developed subcutaneous tumors in the neck and trunk regions from ages ranging from 6 months to 16 months. Tumor formation to date shows a penetrance of 77%. Immunohistochemical analysis characterized the tumors as either UPSs or leimyosarcomas. About 30% of these tumors displayed stabilization of p53, a common event also seen in human sarcomas. We derived two cell lines (KHC-1, KHC-2) from the sarcomas of separate mice, and analyzed the levels of STAT family proteins in these cell lines. Stat3 was noted to be constitutively activated in these sarcoma cell lines. Persistent activation of Stat3 is seen in diverse human malignancies such as breast and head and neck cancers. To determine if restoring Usp18 activity reverses tumorigenicity in these cells, human USP18 was introduced retrovirally. Cells re-expressing USP18 versus empty vector showed a substantial decrease in anchorage independent growth in soft agar and also had reduced levels of activated Stat3 (pStat3). Furthermore, treatment of the parental cell lines with a Stat3 inhibitor, JSI-124, led to decreased cell growth, apoptosis and reduced levels of pStat3. In summary, Usp18 knockout mice spontaneously develop sarcomas, whose tumorigenicity can be partially reversed by reintroduction of Usp18. Thus, this new and tractable model should prove useful to elucidate the activity of Stat3 targeting and other agents to treat sarcomas. Citation Format: Fadzai Chinyengetere, Andrew Giustini, David Sekula, Sarah Freemantle, Vincent A. Memoli, Paul Jack Hoopes, Ethan Dmitrovsky. Spontaneous formation of sarcomas in Usp18 knockout mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2013-1089