Leonard B MaggiJr

Summary: Induction of the MHC class I antigen processing and presentation pathway (C1APP) is a critical part of the IFN-γ response necessary for effective cytotoxic immunity against tumors of epithelial origin ^1,2 . Loss of this response is associated with worse disease outcomes and renders patients refractory to immunotherapies ^3–6 . Without C1APP induction, tumor cells cannot optimally process and present immunopeptides from tumor-associated antigens (TAA) and neoantigens to effector cytotoxic T cells ^7–9 . Here, we show that physiologic levels of hypoxia block induction of the immunoproteasome (IP) and other C1APP components in cancer cells, including human non-small cell lung cancer (NSCLC). In A549 cells, this leads to impaired presentation of more than 73% of detectable immunopeptides, including TAA and neoantigen-derived immunopeptides. This effect is independent of HIF-1α or HIF-2α signaling, protein degradation, autophagy, or stimulus type. Instead, hypoxia induces translational arrest of C1APP mRNAs prior to complete monosome loading, along with sequestration into hypoxia-associated stress granules. This phenomenon is reversible with the epitranscriptomic compound 5-azacytidine. Consistent with these findings, IP expression is excluded from hypoxic regions in most human NSCLC tumors. Together, these results link tumor hypoxia to a state of “immunogenic dormancy” and identify stress granules as a previously unrecognized mechanism of immune escape.

Triple-negative breast cancer (TNBC), lacking expression of estrogen, progesterone, and HER2 receptors, is aggressive and lacks targeted treatment options. Therefore, the mission to identify actionable targets remains the top priority. We recently demonstrated that an RNA editing enzyme, adenosine deaminase acting on RNA 1 (ADAR1), plays important roles in TNBC’s ability to initiate and maintain tumorigenesis. We posit that ADAR1 functions as a homeostatic factor protecting TNBC from stresses when otherwise kept in check, contributing to tumorigenesis. By both targeted and unbiased approaches, we identified two metabolic vulnerabilities, 1) iron-dependent cell death pathway, ferroptosis, and 2) serine addiction, associated with ADAR1 in TNBC. By performing single-reaction monitoring-based liquid chromatography coupled to mass spectrometry (LC-MS) to measure intracellular lipid contents, we showed that ADAR1 loss in TNBC cells increased the abundance of polyunsaturated fatty acid phospholipids (PUFA-PL), of which peroxidation is the primary driver of ferroptosis. Transcriptomic analysis and phenotypic drug screen using a ferroptosis-focused library were performed to identify underlying mechanisms and drug candidates synergizing with ADAR1 loss through ferroptosis, respectively. Meanwhile, an unbiased screen using a FDA-approved compound library revealed that ADAR1 loss also sensitizes TNBC cell line MDA-MB231 to inhibition of the serine biosynthesis pathway. We showed that ADAR1 knockdown leads to altered serine transport, and a metabolic flux analysis using labeled (U-¹³C) glucose will be performed to determine if ADAR1 loss affects de novo serine biosynthesis and one-carbon metabolism. By testing the hypothesis that ADAR1 regulates the metabolic fitness of TNBC by desensitizing ferroptosis and serine addiction, we aim to leverage these metabolic vulnerabilities to inform basic, pre-clinical, and clinical studies to develop novel therapeutic strategies for TNBC. Citation Format: Che-Pei Kung, Leah P Shriver, Nick Terzich, Ma Xenia G Ilagen, Mike Prinsen, Sua Ryu, Raleigh D Kladney, Gary J Patti, Leonard B Maggi, Jason D Weber. ADAR1-associated metabolic vulnerabilities in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A104.

Relationships with mentors and labmates are defining aspects of a researcher's journey in science. Ideally, these interactions are outstanding opportunities to learn from others and provide the basis for lifelong collaborations. Unfortunately, sometimes interpersonal dynamics in the lab are challenging. Graduate students entering the lab can greatly benefit from advice about navigating the interpersonal aspects of doing science. This article covers essential recommendations for developing a good trainee‐mentor relationship and working well with peers in the lab, or being a “good lab citizen.” Lab members–especially graduate students–often spend more time with labmates than with their friends and family during their graduate career, making these relationships essential to their well‐being. The guidance also covers some advice for handling a tense relationship or problematic work environment. Finally, the advice concludes by discussing how to manage the fear of failure, overcome imposter syndrome, develop self‐awareness, and cope with stress. These four issues are fundamental to success in research but are not discussed with graduate students as much as may be necessary. © 2021 Wiley Periodicals LLC.This article was corrected on 25 July 2022. See the end of the full text for details.

Researchers must conduct research responsibly for it to have an impact and to safeguard trust in science. Essential responsibilities of researchers include using rigorous, reproducible research methods, reporting findings in a trustworthy manner, and giving the researchers who contributed appropriate authorship credit. This “how‐to” guide covers strategies and practices for doing reproducible research and being a responsible author. The article also covers how to utilize decision‐making strategies when uncertain about the best way to proceed in a challenging situation. The advice focuses especially on graduate students, but is appropriate for undergraduates and experienced researchers. It begins with an overview of responsible conduct of research, research misconduct, and ethical behavior in the scientific workplace. The takeaway message is that responsible conduct of research requires a thoughtful approach to doing research in order to ensure trustworthy results and conclusions, and that researchers receive fair credit. © 2021 Wiley Periodicals LLC.This article was corrected on 25 July 2022. See the end of the full text for details.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1 knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.

Summary: Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of cellular double-stranded RNAs (dsRNA). ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that ADAR1 expression and editing activity is required in TNBC cell lines but not in ER+ and/or Her2+ cells. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. PKR expression is elevated in TNBC and its activity is induced upon ADAR1-knockdown, which correlates with a decrease in translation. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescues the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

Overexpression of the multiple myeloma set domain ( MMSET ) Wolf‐Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t (4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138+ tumor cells from t (4;14)‐positive and ‐negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t (4;14)‐negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11‐driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid‐derived 2)‐like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11‐driven increases in ROS; however, transcriptional up‐regulation of some of NRF2's antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.—Mahajan, N., Wu, H.‐J., Bennett, R.L., Troche, C., Licht, J.D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA. FASEB J. 31, 482–490 (2017). www.fasebj.org

Individuals with robust natural killer (NK) cell function incur lower rates of malignancies. To expand our understanding of genetic factors contributing to this phenomenon, we analyzed NK cells from cancer resistant and susceptible strains of mice. We identified a correlation between NK levels of the X-chromosome-located adaptor protein SLy1 and immunologic susceptibility to cancer. Unlike the case for T or B lymphocytes, where SLy1 shuttles between the cytoplasm and nucleus to facilitate signal transduction, in NK cells SLy1 functions as a ribosomal protein and is located solely in the cytoplasm. In its absence, ribosomal instability results in p53-mediated NK cell senescence and decreased clearance of malignancies. NK defects are reversible under inflammatory conditions and viral clearance is not impacted by SLy1 deficiency. Our work defines a previously unappreciated X-linked ribosomopathy that results in a specific and subtle NK cell dysfunction leading to immunologic susceptibility to cancer.