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Despite advancements in treatment options, the overall cure and survival rates for non–small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC. Significance: Epigenome-wide CRISPR knockout screens identify NPM1 as a novel metabolic vulnerability and demonstrate that targeting NPM1 is a new therapeutic opportunity for patients with NSCLC.

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (Npm1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC.

The implementation of various mouse models is critical to asses safety, efficacy, and short- and long-term persistence of therapeutic modalities, especially for cell-based therapies. To increase our repertoire of viable humanized murine models, we developed two in vivo models by taking advantage of Taconic's immunodeficient mice, one monitoring graft versus host disease (GvHD) and the other addressing human natural killer (NK) cell cytotoxicity. We utilized NOG mice to develop a model of GvHD, by transplanting human PBMCs at varying doses and monitoring mice for changes in body weight over time. We determined a dose of injected PBMCs that allowed for GvHD to occur yet provided a window of opportunity for potential therapies to slow progression. When human natural T regulatory cells (nTregs) were co-injected with PBMCs in NOG mice, there was prolonged survival and a less rapid loss of body weight as compared to PBMCs alone. To create an NK cytotoxicity model, we transplanted human primary NK cells into NOG-hIL15 mice, which are NOG mice that constitutively produce human IL-15. We showed successful engraftment and proliferation of NK cells, with peak engraftment occurring 4-5 weeks post injection, and that these human primary NK cells were able to persist without signs of xenogeneic GvHD. Utilizing K562 tumor cells that express luciferase, we found these engrafted NK cells have fast and potent cytotoxic activity using IVIS imaging, resulting in elimination of tumor cells as compared to non-engrafted mice. Our results collectively suggest that the two in vivo models developed here will be valuable tools for investigating the clinical benefit of immune cell-based therapeutics. Citation Format: Elizabeth McMichael, Utsav Jetley, Christopher Rudulier, Minasri Borah, Nicole Ganci, Vandhana Ragothaman, Ishina Balwani, Amanda Frain, Priya Pajanirassa, Yuko Miki, Jeffrey Jones, Troy Luster, Marie Keenan, Terina Martinez, Yong Zhang, Birgit Schultes. In vivo model development for immune cell-based therapeutics [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 5574.

The clinical successes achieved by different immunotherapies have resulted in a paradigm shift in treatment modalities. Despite these significant advances, not all patients benefit from the use of these therapies, creating a need to develop additional approaches to enhance and broaden their clinical application. To identify genes whose products can increase or decrease the sensitivity of tumor cells to the immune system, we used a CTL assay to screen a whole genomic CRISPR library. We co-culture a mouse cell line, ID8, expressing a model antigen (Ova) with transgenic CD8 T cells (OT-I) recognizing this antigen. A set of controls that enhance or decrease CTL activity behaved as expected. Comparison of the CRISPR score identified several hits that increased or decreased the sensitivity of the tumor cells to CTL killing. Subsets of these hits belong to two pathways involved in CTL-mediated killing: the IFN-γ and the TNF-α signaling pathways. We evaluated which of these hits would be amenable to therapeutic modulation, and decided to focus on the kinase TAK1 for confirmation and validation studies. A TAK1 deficient cell line was more sensitive to CTL killing, which was prevented by expression of TAK1, confirming the role of TAK1 in this process. A TAK1 gene carrying an inactivation mutation K63W did not rescue the effects of TAK1 KO, indicating that TAK1 enzymatic activity was necessary. Several pathways mediate CTL killing: Perforin/Granzyme B, IFN-γ, TNF-α, Fas & TRAIL pathways. To determine TAK1 MOA, we studied the effects of a Perforin/Granzyme B inhibitor CMA. CMA inhibited CTL activity in a dose-dependent manner on WT cells, but did not inhibit CTL activity on TAK1 deficient cells, indicating TAK1 effects are independent of this pathway. We then tested the sensitivity of TAK1 KO cells to TNF-α. TAK1 KO cells were more sensitive to TNF-α mediated killing, and similar results were observed with several additional cell lines (MC38, EMT6, KP). TNF-α can activate the JNK, p38, and NF-κB pathways, and the apoptosis extrinsic pathway to regulate cell growth and cell death. Kinetics studies monitoring pathway activity upon TNF-α stimulation showed that TAK1 KO cell lines induced cFLIP degradation before observing PARP cleavage, and that the NF-κB pathway, which has been observed to mediate cFLIP synthesis, was not activated. We proceeded to evaluate the effects of TAK1 deficiency in a mouse syngeneic model. TAK1 deficiency resulted in reduced growth and increased survival in the MC38 in vivo model. In summary, by screening a CRISPR library against a CTL assay, we identified TAK1 as a novel potential target for immunotherapies. TAK1 deficiency enhances CTL killing and results in decreased tumor growth and increased survival in vivo. This results support the development of TAK1 inhibitors to enhance the anti-tumor action of the immune system. Citation Format: Juan J. Miret, Troy A. Luster, Patrick Lizotte, Min Wu, Sarah Nzikoba, Luke Taus J. Taus, Prafulla C. Gokhale, Paul Kirschmeier, David Barbie, Cloud P. Paweletz. TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α [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 5543.

As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells. Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing [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 4935.

B lymphocyte stimulator (BLyS) is a member of the TNF superfamily of cytokines. BLyS is expressed by cells of myeloid origin and is one of many soluble factors that regulate human B-cell activation. The biological activity of BLyS is mediated by three cell surface receptors: B-cell-activating factor-receptor (BAFF-R/BR3), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA). These receptors are expressed by normal and malignant B-cells at various stages of development. To target malignant B cells expressing BLyS receptors, the recombinant plant-derived toxin gelonin was fused to the N-terminus of BLyS to generate the fusion toxin BLyS-gel. Gelonin is an N-glycosidase that removes a critical adenine from eukaryotic 28S rRNA, disrupting ribosome function and inhibiting protein synthesis. Importantly, gelonin is not toxic to cells unless coupled with a targeting moiety that can enter the cell. BLyS-gel was used to treat a panel of nearly 50 malignant non-Hodgkin lymphoma (NHL) cell lines expressing BLyS receptors. NHL subtypes mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and B cell precursor-acute lymphocytic leukemia (BCP-ALL) were preferentially sensitive to BLyS-gel mediated cytotoxicity, with EC50 values in the low picomolar range. BLyS-gel cytotoxicity was mediated primarily by BR3 and TACI in sensitive cell lines; however, cell surface expression of these BLyS receptors did not necessarily confer sensitivity to BLyS-gel. The internalization of BLyS-gel was confirmed in both sensitive and resistant cell lines, indicating resistance was not due to a lack of internalization. As expected, BLyS-gel treatment inhibited protein synthesis in sensitive, but not resistant cell lines. In most cell lines, cell death seemed to be mediated by the “ribotoxic stress response.” This response involves activation of p38 MAPK and JNK/SAPK, and BLyS-gel mediated cytotoxicity was inhibited by the p38/JNK inhibitor SB203580. Finally, BLyS-gel treatment significantly prolonged survival in three distinct disseminated xenograft models (BCP-ALL, DLBCL & MCL) in SCID mice, and BLyS-gel was shown to specifically localize to sites of disease. Together, these findings suggest BLyS has potential as a targeting ligand for the therapeutic delivery of toxins and/or drugs directly to malignant B cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3861. doi:1538-7445.AM2012-3861

Bavituximab is a chimeric monoclonal antibody that is being combined with chemotherapy to treat patients with lung or pancreatic cancer in randomized Phase II clinical trials. Bavituximab targets the immunosuppressive lipid, phosphatidylserine (PS), which becomes exposed on the outer membrane surface of tumor blood vessels and tumor cells in tumors responding to therapy. The antibody acts by destroying tumor vasculature and by reactivating tumor immunity. Here, we generated new PS-targeting therapeutics by fusing domains of the PS-binding plasma protein, mouse β2-glycoprotein I (≥2GP1), to the Fc region of mouse IgG2a. Such ‘betabodies’ potentially have advantages over bavituximab. They bind directly to PS, whereas bavituximab requires a cofactor protein (≥2GP1) for binding; they can be made fully human; they are smaller in size (100KDa versus 250KDa for the bavituximab: β2GP1 complexes); and they have slower blood clearance rates. Many different constructions of betabodies were tested, each having different orientations, domains, and glycosylation patterns. Constructs were identified that bound strongly to PS-expressing cells and plates, localized to tumor vascular endothelium in vivo, and had α-phase blood half-lives of approximately seven days after intravenous injection into mice as compared with two days for a murine version of bavituximab. Betabodies could potentially be the next generation of PS-targeting cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4632. doi:1538-7445.AM2012-4632