Faculty Bibliography

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Molecular virology tools are critical for basic studies of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. There remains a need for experimental systems that do not rely on viruses capable of spread that could potentially be used in lower containment settings. Here, we develop spike-deleted SARS-CoV-2 self-replicating RNAs using a yeast-based reverse genetics system. These non-infectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate Replicon Delivery Particles (RDPs) for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and characterizing viral variants.

Background: Small cell lung cancer (SCLC) is an exceptionally aggressive disease comprising 13% of all lung cancer cases. With limited treatment options that typically result in transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The recent addition of immunotherapy to first-line platinum-based doublet chemotherapy shows only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second-line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need.
Method(s): To identify targets sensitizing to chemotherapy we performed an in vitro CRISPR screen in SCLC cell lines from all major SCLC subtypes. Candidate hits were validated genetically, and pharmacologically with in vitro synergy assays and PDX treatments. Signaling pathways were studied by western blot, and toxicity studies were performed in vivo, to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs).
Result(s): Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a contributor to chemotherapy resistance in all SCLC subtypes. Combination of selinexor, an Exportin 1 inhibitor approved for clinical use in hematological malignancies, with cisplatin or irinotecan demonstrated synergy in vitro and exquisite efficacy in vivo in chemonaive and chemoresistant SCLC PDXs representing all SCLC subtypes. This efficacy was associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation. The combinations were well tolerated in mice. We found SCLC to have the highest XPO1 mRNA expression among a diverse array of tumor histologies, which was confirmed at the protein level in clinical TMAs.
Conclusion(s): Exportin 1 inhibition enhances sensitivity to the chemotherapeutic drugs used in first-line and second-line treatment of SCLC tumors. Our results provide preclinical rationale for the combination of selinexor with cisplatin or irinotecan in naive and relapsed SCLC, respectively. Legal entity responsible for the study: The authors.
Funding(s): Supported by NCI R01 CA197936 and U24 CA213274 (CMR), the Druckenmiller Center for Lung Cancer Research (CMR, TS, AQV), NIH K08 CA-248723 (AC) and the Van Andel Institute - Stand Up to Cancer Epigenetics Dream Team grant (CMR). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. We acknowledge the use of the Integrated Genomics Operation Core, funded by the NCI Cancer Center Support Grant (CCSG, P30 CA08748), Cycle for Survival, and the Marie-Josee and Henry R. Kravis Center for Molecular Oncology. The Precision Pathology Center is supported by the NCI Cancer Center Support Grant P30-CA008748. Disclosure: A. Quintanal-Villalonga: Financial Interests, Personal, Invited Speaker: AstraZeneca. M. Offin: Financial Interests, Personal, Other: PharmaMar; Financial Interests, Personal, Other: Novartis; Financial Interests, Personal, Other: Targeted Oncology; Financial Interests, Personal, Other: Bristol Myers Squib; Financial Interests, Personal, Other: Merck Sharp & Dohme. C.M. Rudin: Financial Interests, Personal, Other: AbbVie; Financial Interests, Personal, Other: Amgen; Financial Interests, Personal, Other: Ascentage; Financial Interests, Personal, Other: AstraZeneca; Financial Interests, Personal, Other: Bicycle; Financial Interests, Personal, Other: Celgene; Financial Interests, Personal, Other: Daiichi Sankyo; Financial Interests, Personal, Other: Genentech/Roche; Financial Interests, Personal, Other: Ipsen; Financial Interests, Personal, Other: Jazz; Financial Interests, Personal, Other: Lilly; Financial Interests, Personal, Other: Pfizer; Financial Interests, Personal, Other: PharmaMar; Financial Interests, Personal, Other: Syros; Financial Interests, Personal, Other: Vavotek; Financial Interests, Personal, Advisory Board: Bridge Medicines; Financial Interests, Personal, Advisory Board: Earli; Financial Interests, Personal, Advisory Board: Harpoon Therapeutics. All other authors have declared no conflicts of interest.
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Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted ⁸⁹Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of ⁸⁹Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting ⁸⁹Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, ⁸⁹Zr-DFO_PODS-^DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to ⁸⁹Zr-DFO_Mal-^DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a ⁸⁹Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited ∼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.

Immune checkpoint blockade (ICB) has been a remarkable clinical advance for cancer; however, the majority of patients do not respond to ICB therapy. We show that metastatic disease in the pleural and peritoneal cavities is associated with poor clinical outcomes after ICB therapy. Cavity-resident macrophages express high levels of Tim-4, a receptor for phosphatidylserine (PS), and this is associated with reduced numbers of CD8⁺ T cells with tumor-reactive features in pleural effusions and peritoneal ascites from patients with cancer. We mechanistically demonstrate that viable and cytotoxic anti-tumor CD8⁺ T cells upregulate PS and this renders them susceptible to sequestration away from tumor targets and proliferation suppression by Tim-4⁺ macrophages. Tim-4 blockade abrogates this sequestration and proliferation suppression and enhances anti-tumor efficacy in models of anti-PD-1 therapy and adoptive T cell therapy in mice. Thus, Tim-4⁺ cavity-resident macrophages limit the efficacy of immunotherapies in these microenvironments.

This standardized protocol describes the preparation of PCR amplified and purified samples from human cell lines passaged and collected from CRISPR screening. High-quality samples can be used to perform next-generation sequencing (NGS) to uncover changes in sgRNA abundance from the timepoint at which library-transduced cells are selected to the timepoint when the screen is ended. Here, we describe proper calculation methods for library representation and show how to overcome potential issues often encountered by researchers. For complete information on the use and execution of this protocol, please refer to Wohlhieter et al. (2020).

Seneca Valley virus (SVV) is a picornavirus with potency in selectively infecting and lysing cancerous cells. The cellular receptor for SVV mediating the selective tropism for tumors is anthrax toxin receptor 1 (ANTXR1), a type I transmembrane protein expressed in tumors. Similar to other mammalian receptors, ANTXR1 has been shown to harbor N-linked glycosylation sites in its extracellular vWA domain. However, the exact role of ANTXR1 glycosylation on SVV attachment and cellular entry was unknown. Here we show that N-linked glycosylation in the ANTXR1 vWA domain is necessary for SVV attachment and entry. In our study, tandem mass spectrometry analysis of recombinant ANTXR1-Fc revealed the presence of complex glycans at N166, N184 in the vWA domain, and N81 in the Fc domain. Symmetry-expanded cryo-EM reconstruction of SVV-ANTXR1-Fc further validated the presence of N166 and N184 in the vWA domain. Cell blocking, co-immunoprecipitation, and plaque formation assays confirmed that deglycosylation of ANTXR1 prevents SVV attachment and subsequent entry. Overall, our results identified N-glycosylation in ANTXR1 as a necessary post-translational modification for establishing stable interactions with SVV. We anticipate our findings will aid in selecting patients for future cancer therapeutics, where screening for both ANTXR1 and its glycosylation could lead to an improved outcome from SVV therapy.

Background: A minority of small cell lung carcinomas (SCLC) has minimal or absent expression of neuroendocrine (NE) markers, which can present a diagnostic challenge. Recent studies have suggested that POU2F3-a marker of chemosensory tuft cell lineage-is expressed specifically in NE-low SCLC. Here, we aimed to examine expression of POU2F3 in SCLC with extremely low or negative NE markers and to determine its specificity relative to non-NE lung carcinomas.
Design(s): POU2F3 expression was examined immunohistochemically in 152 SCLC and 116 non-small cell lung carcinomas (NSCLC; 53 adenocarcinomas, 63 squamous cell carcinomas). SCLC comprised 144 unselected cases and 8 additional pre-selected NE-minimal or negative SCLC. All SCLC were tested for 4 conventional NE markers (CNM; synaptophysin, chromogranin, CD56, and INSM1), and tumors with combined NE score (average H-score of 4 CNM) <50 were defined as NE-low and those with score <10 (staining isolated cells only) as NE-minimal. TTF-1 expression was also evaluated.
Result(s): POU2F3 was expressed in 8% of unselected SCLC (11/140), but was completely negative in all 116 NSCLC. In the whole cohort, compared to POU2F3-negative cases (n=134), POU2F3-positive SCLC (n=18) had fewer positive CNM (mean 1.8 vs 3.7, p<0.0001), lower combined NE score (mean 60 vs 183, p<0.0001) and lower rate of TTF-1 expression (6% vs 81%, p<0.0001), respectively. A total of 15 SCLC were NE-low (n=10), NEminimal (n=4) or NE-entirely negative (n=1). POU2F3 was positive in 10/15 (67%) of these cases, including all 5 NE-minimal/negative SCLC. POU2F3 expression in these cases was typically strong and diffuse (mean H-score 147; range 60-235).
Conclusion(s): POU2F3 expression is highly specific for SCLC relative to NSCLC, and is significantly enriched in NE-low SCLC, particularly in cases with minimal or negative NE marker expression. We suggest that POU2F3 represents a novel helpful diagnostic marker of SCLC