bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1179: MSH2 is necessary for temozolomide-induced ATR activation in MGMT-methylated cancers
Authors: Ganesa Sachita , Sule Amrita D. , Jackson Christopher B. , Sundaram Ranjini K. , Bindra Ranjit S.
Published: July 2021

The methylation status of the O6-methyl guanine methyltransferase (MGMT) gene promoter is a prognostic biomarker for treatment with the alkylator, temozolomide (TMZ) in many solid tumors including gliomas and colorectal cancers. It is well established that patients with a methylated MGMT promoter (MGMT-) who are treated with the TMZ have a better overall survival than patients with an unmethylated MGMT promoter (MGMT+). The enzyme produced by the MGMT gene is responsible for removing cytotoxic O6-methylguanine (O6-meG) lesions formed by TMZ. In the MGMT- setting, the O6-meG lesion activates the mismatch repair (MMR) pathway which functions to remove the damage. Published work from our group reported differential activation of the ataxia telangiectasia and RAD3 related protein (ATR) in MGMT- and MGMT+ glioblastoma multiforme (GBM) cells in response to TMZ treatment, as demonstrated through the phosphorylation of CHK1. Though it is known that MMR proteins are involved in ATR activation, the specific MMR proteins required for ATR activation by TMZ-induced alkyl lesions remain unknown in the MGMT- setting. Here, we demonstrate that the MMR protein MSH2 plays a role in ATR activation in the presence of O6-meG lesions. We show that there is potent synergy with ATRi and TMZ in the MGMT- MSH2-proficient GBM cell line, which is abrogated in an shMSH2 MGMT- GBM cell line. Additionally, we observe decreased levels of pCHK1 in the shMSH2 MGMT- setting compared to the MGMT- MSH2-proficient cells, suggesting that MSH2 is integral in the activation of ATR upon TMZ treatment. This study elucidates a potential role for MSH2 in ATR activation. Mechanistic understanding of how the MMR system is involved in ATR activation by TMZ can ultimately be exploited for therapeutic gain.

Citation Format: Sachita Ganesa, Amrita D. Sule, Christopher B. Jackson, Ranjini K. Sundaram, Ranjit S. Bindra. MSH2 is necessary for temozolomide-induced ATR activation in MGMT-methylated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1179.
Abstract 1474: Antitumor activity of belvarafenib in melanoma brain metastasis and NRAS mutation melanoma models
Authors: Bae Inhwan , Kim Yu-Yon , Kim Jisook , Park Hyunjin , Song Taehyun , Byun Joo-Yun , Lim Semi , Kim Young Hoon , Ahn Young Gil , Suh Kwee Hyun
Published: July 2021

Over the past decade, BRAF inhibitors, either alone or in combination with MEK inhibitors, have set a new milestone in the treatment of patients with BRAF V600 mutation melanoma, which accounts for about 50% of melanomas. More recently, anti-PD-(L)1 therapies such as nivolumab or pembrolizumab and atezolizumab have been approved as very useful treatments for patients with melanoma. Nevertheless, metastases to the brain in melanoma patients, which are highly aggressive and associated with poor outcomes, still require better treatment. Also, to date, no targeted therapy has been developed for patients with NRAS mutations, which account for about 20% of melanoma.

Previously, we reported that belvarafenib (HM95573 or GDC5573) strongly inhibits the growth of various cancer cell lines harboring BRAF, NRAS or KRAS mutations and demonstrates remarkable anticancer efficacy in preclinical animal models. Belvarafenib, currently in Phase 1 clinical trials, is a potent and selective pan-RAF kinase inhibitor showing strong inhibition activities for BRAF WT, BRAF V600E and CRAF.

Here, we present the results of preclinical model studies suggesting that belvarafenib could be effective in treating melanoma patients with metastases to the brain as well as NRAS mutation melanoma patients. First, belvarafenib was found to be able to accumulate in the brain of mice and rats following oral administration. The exposure of belvarafenib in the brain was similar to that in plasma (approximately 100% brain to plasma ratio). This level of brain penetration of belvarafenib differs significantly from currently approved BRAF inhibitors that are known to have low brain penetration. Belvarafenib showed excellent anti-tumor activity in an orthotopic brain tumor model using melanoma cells. Belvarafenib significantly increased the overall survival of mice implanted intracranially with BRAF V600E A375SM melanoma cells. Overall, our data demonstrated that belvarafenib has therapeutic potential to treat NRAS mutation melanoma patients. In fact, in the NRAS G13D K1735 murine melanoma cell syngeneic mice model, the combination of belvarafenib with atezolizumab (anti-PD-L1) significantly inhibited tumor growth and effectively induced infiltration of cytotoxic T-cells into tumor tissues. These effects of belvarafenib identified in the above preclinical studies need to be immediately warranted through appropriate clinical trials.

Citation Format: Inhwan Bae, Yu-Yon Kim, Jisook Kim, Hyunjin Park, Taehyun Song, Joo-Yun Byun, Semi Lim, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. Antitumor activity of belvarafenib in melanoma brain metastasis and NRAS mutation melanoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1474.
Abstract 1525: Enhancing potency of antigen presenting cells via signal 2/3 mRNA engineering through Cell Squeeze® technology
Authors: Ozay Emrah Ilker , Dunbar Paul , Volk Kelly , Maloney Michael F. , Yee Christian , Mosaheb Mubeen , Trumpfheller Christine , Umana Pablo , Seidl Katherine J. , Bernstein Howard , Loughhead Scott M.
Published: July 2021

Antigen-specific CD8+ T cells are critical effectors of the immune system that help limit persistence of tumors and virally infected cells. During priming, T cells integrate three signals which determine the magnitude and quality of response generated. Signal one is T cell receptor(TCR) and peptide-MHC (pMHC) engagement, which determines the specificity of the response.Signal two is cell surface costimulation by antigen presenting cells (APCs). Signal three is provided through the local cytokine milieu at the time of priming. Here, we use microfluidic CellSqueeze® technology to deliver mRNAs encoding antigen (signal 1), costimulatory molecules(signal 2), and chimeric membrane-tethered cytokines (signal 3) to the cytosol of human peripheral blood mononuclear cells (PBMCs), generating antigen presenting cells (APCs) with multiple enhanced functions. We demonstrate that microfluidic squeezing enables delivery and expression of single or multiple mRNAs encoding signal 1 (various antigens), signal 2 (CD70 orCD86) and/or signal 3 (membrane-tethered form of IL-2) by the major subsets of PBMCs (T cells, B cells, NK cells, and monocytes). While unsqueezed PBMCs showed no to minimal expression of signal 2 and no expression of signal 3 molecules, expression of delivered signal 2and 3 mRNAs in squeezed PBMCs could be observed on the cell surface for several days post squeeze delivery - a timeframe that could potentially support improved T cell priming. When these signal 2/3 molecules were delivered alone or in combination, antigen-specific CD8+ T cell responses could be increased as much as ten-fold compared to delivery of antigen alone.Therefore, microfluidic cell squeezing enables us to efficiently deliver mRNA antigens that have potential to generate multiple immune epitopes in an HLA agnostic manner. Moreover,multiplexing these antigens with signal 2/3 mRNAs enhances the antigen presenting potency ofSQZ APCs inducing stronger antigen-specific CD8+ T cell responses. The potential to deliver numerous materials simultaneously and engineer compound signals via mRNA could allow for applications for HLA-agnostic patient population in oncology and infectious disease areas.

Citation Format: Emrah Ilker Ozay, Paul Dunbar, Kelly Volk, Michael F. Maloney, Christian Yee, Mubeen Mosaheb, Christine Trumpfheller, Pablo Umana, Katherine J. Seidl, Howard Bernstein, Scott M. Loughhead. Enhancing potency of antigen presenting cells via signal 2/3 mRNA engineering through Cell Squeeze® technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1525.
Abstract 1593: Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy
Authors: Deronic Adnan , Thagesson Mia , Nilsson Anneli , Ellmark Peter , Fält Anette , Fält Charlotte , Smith Karin Enell
Published: July 2021

Mitazalimab (ADC-1013, JNJ-64457107) is a human CD40 agonistic antibody developed for immunotherapy of cancer. Activation of CD40, expressed on antigen-presenting cells (APC), leads to improved T cell priming and initiation of T cell-dependent anti-tumor responses. There are several CD40 agonists in clinical development with different characteristics, affecting both efficacy and tolerability. Mitazalimab is an FcγR crosslinking-dependent IgG1 antibody, with potential for high efficacy and safety. Immunologically “cold” tumors, such as pancreatic cancer, are defined by low infiltration of immune cells as well as low expression and release of neoantigens. In pancreatic cancer, infiltration of effector CD8+ T cells is blocked by the desmoplastic tumor stroma, which surrounds the tumor and hosts immune-suppressive macrophages that dampen the immune response in the tumor microenvironment (TME). By activating and re-directing the immunosuppressive M2 macrophages into tumoricidal M1 macrophages in the TME, CD40 agonists such as mitazalimab, have the potential to augment the response to chemotherapy and spark an effective immune reaction by i) priming T cells reactive to the tumor neoantigens released by the chemotherapy, and ii) inducing degradation of the stroma surrounding the tumor enhancing the efficacy of chemotherapy. The ability of mitazalimab to augment the response to chemotherapy was demonstrated in mice, transgenic for human CD40 (hCD40tg), inoculated with the syngeneic tumor cell line MB49. Mitazalimab, administered repeatedly together with FOLFIRINOX (oxaliplatin, irinotecan, 5-fluorouracil and folinic acid), synergized effectively with chemotherapy and induced a long-term survival. Further, it was also demonstrated that mitazalimab induced a concentration-dependent activation of tumor-associated macrophages (TAMs), purified from human tumor samples, into a more tumoricidal M1 like phenotype, by upregulation of cell surface markers such as CD83.In conclusion, mitazalimab re-directs human macrophages into a M1 like tumoricidal, less immunosuppressive phenotype, and synergizes effectively with chemotherapy, leading to induction of a long-term survival in a preclinical tumor mouse model. These preclinical data, together with the clinical data of mitazalimab from the phase 1 study (NCT02829099) where mitazalimab was well tolerated up to 1200 μg/kg with manageable side effects, support further clinical development of mitazalimab in combination with chemotherapy in pancreatic cancer.

Citation Format: Adnan Deronic, Mia Thagesson, Anneli Nilsson, Peter Ellmark, Anette Fält, Charlotte Fält, Karin Enell Smith. Mitazalimab, a potent CD40 agonist with potential for combination with chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1593.
Abstract 1611: A novel anti-human CD47 antibody prodrug as cancer therapeutics to lower on-target side effects
Authors: Zhang Richard , Drobenaire Hunter , Li Huey , Pizzarella Veronica , Lin Haishan
Published: July 2021

CD47 is highly expressed in many types of cancers, including human acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and solid tumors such as small-cell lung cancer (SCLC). Binding of CD47 to its receptor, SIRPα, on macrophages leads to inhibition of macrophage activation and phagocytosis. CD47 overexpression is one of the mechanisms cancer cells employ to evade immune surveillance and correlates with poor prognosis in cancers. Several strategies have been used to block CD47 binding to SIRPα, including anti-CD47 blocking antibodies and SIRPα-Fc fusion proteins. However, since CD47 is also expressed on normal cells, a major concern is that anti-CD47 antibodies could also cause on-target side effects, such as red blood cell hemagglutination and anemia. We have identified a panel of high affinity anti-human CD47 blocking antibodies. The lead antibodies bound to human CD47 with sub-nanomolar affinities and displayed a range of activities in blocking CD47:SIRPα interaction. These antibodies showed robust activities in stimulating macrophage-mediated phagocytosis of cancer cells. To minimize the on-target side effects of anti-CD47 antibodies, we have taken an antibody prodrug approach in which the antibody is inactive for normal cells while it becomes functional in the tumor microenvironment enriched with proteases. By screening a phage peptide library, we identified peptides that specifically block the anti-CD47 antibody binding activity. These peptides were fused to the humanized anti-CD47 antibody via a linker containing protease substrate sequences to derive the anti-CD47 antibody prodrug. We demonstrated that the anti-CD47 antibody prodrug lacked CD47 binding activity and was unable to block CD47:SIRPα interaction. Upon cleavage of the anti-CD47 antibody prodrug with proteases, the antibody regained full activities in both CD47 binding and blocking CD47:SIRPα interaction. Importantly, we showed that the anti-CD47 antibody prodrug had minimal hemagglutination and RBC binding activities. These data suggest that the novel anti-CD47 antibody prodrug is a promising candidate for the next generation anti-CD47 therapeutics with improved safety profile.

Citation Format: Richard Zhang, Hunter Drobenaire, Huey Li, Veronica Pizzarella, Haishan Lin. A novel anti-human CD47 antibody prodrug as cancer therapeutics to lower on-target side effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1611.
Abstract 1649: Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model
Authors: Ciccone David , Lazari Vad , Linney Ian , Briggs Michael , Carreiro Samantha , Loh Christine , Tummino Peter , McElwee Joshua , Collis Alan , Kaila Neelu
Published: July 2021

Background: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in negatively regulating signal transduction cascades in cells of hematopoietic origin. Recent data generated using potent and highly selective HPK1 inhibitors underscores the role of HPK1 in negatively regulating T, B, and dendritic cell activation and thus orchestrating a comprehensive anti-tumor immune response. In the present study, we seek to further understand the biological effects of HPK1 inhibition in primary human T cells and in syngeneic models.

Methods: RNA sequencing and multiplex cytokine analyses were utilized to profile both primary human T cells and the MC38 syngeneic model. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models.

Results: In vitro, HPK1 small molecule inhibition resulted in enhanced cytokine production in primary human T cells. In vivo, HPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model.

Conclusion: Pharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity.

Citation Format: David Ciccone, Vad Lazari, Ian Linney, Michael Briggs, Samantha Carreiro, Christine Loh, Peter Tummino, Joshua McElwee, Alan Collis, Neelu Kaila. Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1649.
Abstract 169: Ephesus - A curated content knowledgebase for the clinical interpretation of genomic variants
Authors: Li Jian , Niu Lili , Kinshuk Fnu , Chu Bernie , Mu John , Greene Nicole , Douglas Vivian , Schartner Caitlin , Kannan Kalpana , Rao Priyanka , Krishna Shuba
Published: July 2021

Accurate and comprehensive interpretation of genomic variants has become a bottleneck in clinical sequencing applications due to the implementation of precision oncology and the growth of biomedical findings. We therefore were motivated to build Ephesus, a framework enabling curation of predictive, prognostic and diagnostic evidence for clinical biomarkers in somatic cancers. Currently Ephesus is the primary content source for Roche NAVIFY Mutation Profiler (RUO). The Ephesus' data model ensures adherence to best practices in the field of clinical genomic content curation. Somatic variant classification followed the AMP guidelines. Approvals and recommendations from regulatory agencies and clinical practice guidelines were applied on a regional basis. The data model is mapped to a set of relational tables. Strategies such as data normalization according to standard ontologies, a submit-review-approval workflow with change logs, and biologically relevant data constraints are adopted to enforce data integrity. Ephesus is deployed as a web application whose UI allows users to conduct edits, sorting, filtering, and bulk operations on entities attributes. In order to minimize the manual effort and maximize content coverage, a number of inference rules are applied before the content is ingested into NMP. Currently Ephesus is developed to guide data collection, browsing and summarizing related to primary entities such as Biomarkers, Evidence items, Genes, Variants, Variant groups, and Drugs. The content from 10/28/20 snapshot contains 233k biomarker profiles (including biomarker combinations), in the context of 17 major cancer types. To evaluate the clinical reporting value of the curated knowledge, ~60k clinical cancer patient samples from the AACR GENIE project were queried against Ephesus. Compared our results against those from 3 other major knowledgebases (snapshotted at the same time as Ephesus), we see the performance of Ephesus/NMP exceeds all these knowledgebases.

Patients% with at least one interpretation (numbers in parenthesis include matching by inferences)KnowledgebaseVariantVariant & diseaseTier I variant & diseaseEphesus53 (91)40 (89)19 (25)CGI3573CIViC402215ClinVar452313

Citation Format: Jian Li, Lili Niu, Fnu Kinshuk, Bernie Chu, John Mu, Nicole Greene, Vivian Douglas, Caitlin Schartner, Kalpana Kannan, Priyanka Rao, Shuba Krishna. Ephesus - A curated content knowledgebase for the clinical interpretation of genomic variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 169.
Abstract 1723: WTX-124 is an IL-2 pro-drug conditionally activated in tumors and able to induce complete regressions in mouse tumor models
Authors: Salmeron Andres , Brodkin Heather , Hicklin Daniel , Ismail Nesreen , Morris Kristin , Nirschl Christopher , Seidel-Dugan Cynthia , Steiner Philipp , Steuert Zoe , Sullivan Jenna , Winston William
Published: July 2021

Cancer Immunotherapy has established itself as the fourth pillar of cancer treatment thanks to the clinical success of checkpoint inhibitors. Despite the durable responses achieved by some patients using these new therapies, the proportion of responders remains low and restricted to some cancer types. Preclinical and clinical studies have demonstrated the promise of cytokine therapy to increase antitumor immunity, however systemic toxicity and poor pharmacokinetic profiles have limited their clinical application. One of these key cytokines, interleukin-2 (IL-2), is approved for clinical use in metastatic melanoma and renal cell carcinoma. Unfortunately, this treatment is linked to serious toxicities which limits its utility. To address these limitations, our approach takes advantage of the dysregulated protease tumor microenvironment (TME) to activate an IL-2 pro-drug (IL-2 INDUKINETM) only at the desired site of activity. Peripheral inactivation is achieved by linking the cytokine to an inactivation domain using a tumor protease-sensitive linker. The INDUKINE is also engineered with a half-life extension element to improve tumor exposure. Once the IL-2 INDUKINETM reaches the tumor, tumor-associated proteases cleave the linker and release the active cytokine. This work presents data summarizing the discovery, biochemical, cellular, and in vivo activity of our lead IL-2 INDUKINETM WTX-124 and reviews the mechanism of action of these molecules. Treatment with WTX-124 results in complete regression of tumors driven by the expansion and activation of CD8+ T cells and NK cells which increases the production of effector cytokines in the tumor. Activity is dependent on the proteolytic processing of the INDUKINE as a non-cleavable control INDUKINE lacks efficacy. PK analysis in mouse models demonstrates a favorable accumulation of free IL-2 in tumors compared to plasma. The animals that rejected the tumors upon IL-2 INDUKINETM treatment were protected against follow up re-challenge with the same tumor cell line. In vitro proteolytic activation by human tumor samples will also be presented. The tumor-selective activation of WTX-124 with reduced peripheral toxicities supports its further clinical development.

Citation Format: Andres Salmeron, Heather Brodkin, Daniel Hicklin, Nesreen Ismail, Kristin Morris, Christopher Nirschl, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, Jenna Sullivan, William Winston. WTX-124 is an IL-2 pro-drug conditionally activated in tumors and able to induce complete regressions in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1723.
Abstract 1869: Novel TNFR2 antibodies to overcome T cell exhaustion and suppressive tumor microenvironment
Authors: Sum Chi Shing , Danton Makenzie , hu Qii , Pritsker Alla , Lin Ray , Yu Raymond , Chen Kevin , Tang Fangqiang , Pohl Thomas , Wallner Samantha , Hassan Ahmed , Lu Huarui , Huang Haichun , Pei James , Liu Zhong , Li Han , Lei Ming
Published: July 2021

TNFR2 is highly expressed on the surface of activated T effector cells, T regulatory cells and myeloid cells, and it plays essential roles in modulating the tumor microenvironment. In most cancer types, higher TNFR2 expression correlates with increased immune cell infiltration. Furthermore, the TNFR2 gene is differentially expressed in various cancer types, including breast, lung, renal, liver and skin cancers. In cancers such as head and neck cancer and cutaneous melanoma, high TNFR2 expression correlates with better overall survival. The therapeutic potential of antibodies targeting TNFR2 has been demonstrated by agonist antibodies for the T effector cells, and antibodies that antagonize or deplete the T regulatory cells and suppressive myeloid cells. We discovered novel TNFR2 antibodies that demonstrate unique mechanisms to overcome T cell exhaustion and the suppressive tumor microenvironment for more effective immunotherapy. In vitro studies have shown that our antibodies block TNFα ligand binding and potently inhibit TNFR2 signaling in the monocytic cells. Moreover, these antibodies enhance CD8 T cell function to overcome the suppressive effect from the T regulatory cells and can invigorate exhausted CD8 T cells in an FcγR-dependent manner. In a humanized mouse model, our antibodies demonstrate strong anti-tumor efficacy as single agents or in combination with a PD-L1 inhibitor. Therefore, these antibodies offer potential advantages when the tumor microenvironment is enriched in the exhausted T cells, the suppressive myeloid cells, or the regulatory T cells, as found in anti-PD-1/PD-L1 resistant and PD-1 refractory patients. In conclusion, the data obtained indicate that our TNFR2 antibodies are a novel and promising class of drug candidates for cancer immunotherapeutics. Our lead antibody is currently at the IND-enabling stage with the target of entering clinical studies in early 2022.

Citation Format: Chi Shing Sum, Makenzie Danton, Qii hu, Alla Pritsker, Ray Lin, Raymond Yu, Kevin Chen, Fangqiang Tang, Thomas Pohl, Samantha Wallner, Ahmed Hassan, Huarui Lu, Haichun Huang, James Pei, Zhong Liu, Han Li, Ming Lei. Novel TNFR2 antibodies to overcome T cell exhaustion and suppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1869.
Abstract 1905: In vivo CRISPR screen identifies tumor suppressors as drivers of tumor cell-intrinsic immune evasion
Authors: Min Chengyin , Skoulidis Ferdinandos , Zhou Wenrong , Tsoi Jennifer , Huang Alan , Goldstein Matthew J.
Published: July 2021

Cancer cell-intrinsic genetic alterations that allow cancer cells to evade destruction by the immune system remain poorly characterized. Here we performed a pooled CRISPR-Cas9-based in vivo genetic screen targeted to a pre-defined set of tumor suppressor genes to mimic loss-of-function mutations in syngeneic tumor models. CRISPR edited tumor cells were implanted into immune-deficient or immune-competent C57BL/6 mice, a subset of which were treated with anti-PD1 to simulate increased immune pressure. Tumor samples at the endpoint were subjected to next generation sequencing analysis to identify tumor suppressor genes driving immune evasion. The screen confirmed previously identified immunotherapy targets such as CD47 and Adar as well as known drivers of immune resistance in the interferon signaling and antigen presentation pathways. Importantly, we identified loss of STK11/LKB1 and KEAP1 as drivers of immune resistance in syngeneic models. TIL profiling analysis suggests that STK11/LKB1 knockout induces a “cold” tumor microenvironment. STK11/LKB1 and/or KEAP1 genomic alterations are found in ~25% of non-squamous non-small cell lung cancer and have been reported as a major predictor of primary resistance to PD-1 blockade. Together, these data demonstrate that high-throughput in vivo genetic screens can identify tumor cell-intrinsic drivers of immune evasion and establish murine system relevant to the study of primary resistance to PD-1 axis immunotherapy and more generally, tumor cell-intrinsic immune evasion.

Citation Format: Chengyin Min, Ferdinandos Skoulidis, Wenrong Zhou, Jennifer Tsoi, Alan Huang, Matthew J. Goldstein. In vivo CRISPR screen identifies tumor suppressors as drivers of tumor cell-intrinsic immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1905.
Abstract 1920: Armed oncolytic myxoma virus demonstrates transgene production, function, and therapeutic activity xenograft models
Authors: Franco Lina S. , Abrantes Mario , Abdullahi Wazir , de Matos Ana L. , Walker Benjamin S. , Tacner Zachary , Kien Cassandra , Grigaitis Nicole , McFadden Grant , Potts Steven J. , Sharp Leslie L.
Published: July 2021

Oncolytic viruses (OV) selectively replicate in and lyse tumor cells and provide stimulation to the immune system, representing a promising therapeutic option in development to treat cancers that do not respond well to treatment with immune checkpoint inhibitors. Myxoma virus (MYXV) is a member of the Pox family of double stranded DNA viruses. The natural host of MYXV is a subset of rabbits and hares, but MYXV is able to infect cancer cell lines of humans and other species. The genome of MYXV is relatively large and is amenable to engineering for expression of transgenic proteins, making it an excellent oncolytic virus for introduction of immunomodulatory proteins. Viral reproduction rates, transgene production, transgene function, and inhibition of cell growth of multi-armed myxoma virus was evaluated in vitro using cell lines representing multiple human cancer indications. Efficacy and mechanism of action of multi-armed myxoma virus was also demonstrated in vivo via xenograft models. Multi-armed myxoma represents a novel, engineerable, oncolytic virus with preclinical characteristics that warrant further investigation as an immunomodulatory cancer therapeutic.

Citation Format: Lina S. Franco, Mario Abrantes, Wazir Abdullahi, Ana L. de Matos, Benjamin S. Walker, Zachary Tacner, Cassandra Kien, Nicole Grigaitis, Grant McFadden, Steven J. Potts, Leslie L. Sharp. Armed oncolytic myxoma virus demonstrates transgene production, function, and therapeutic activity xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1920.
Abstract 1962: Voruciclib, a CDK9 inhibitor, downregulates MYC and inhibits proliferation of KRAS mutant cancers in preclinical models
Authors: Wiley Sandra E. , Su Yongwei , Ge Yubin
Published: July 2021

Mutations in KRAS at G12, G13, and Q61 are oncogenic drivers in many cancers, including lung, colorectal, pancreatic, multiple myeloma, and uterine carcinomas. KRAS mutations are frequently accompanied by stabilization of the MYC oncoprotein through increased MYC transcription and decreased protein degradation that is mediated by phosphorylation of MYC on Ser 62 by ERK and CDK9 kinases. Voruciclib is a novel oral inhibitor of CDKs 9, 4, 6, and 1 that is currently being tested in Phase 1B clinical trials for B-cell malignancies and acute myeloid leukemia. Voruciclib inhibition of CDK9 leads to decreased expression of transcriptional targets of RNA Pol II, such as Mcl1 and MYC. To test whether voruciclib could be effective in cancers driven by dysregulated KRAS-MYC signaling, ~20 cancer cell lines with KRAS mutations (G12A, G12C, G12D, G12S, G12V, G13C, G13D, Q61H) were treated in preclinical studies with voruciclib. MTS and Cell Titer Glo assays were used to monitor growth in vitro. Voruciclib decreased viability in all cell lines tested. To investigate MYC protein stability, MIA PACA-2 (G12C) cells were treated with 4 µM voruciclib for 5-240 min, followed by SDS-PAGE and Western Blotting analysis with α-MYC and α-pSer62-MYC antibodies. Voruciclib treatment resulted in a reduction in phosphorylation of MYC on Ser 62. A 60% decrease in pSer62 was observed after 5 min that reached 80% by 60 min. In contrast, there was no decrease in total MYC protein at either 5 or 15 min. A 10% reduction in total MYC was observed at 60 min that reached 50% at 240 min. The ability of voruciclib to inhibit tumor growth in vivo was also tested in murine xenograft models. KRAS mutant human cancer cells HCT-116 (CRC, KRAS G13D), SW-480 (CRC, KRAS G12V), and H-460 (NSCLC, KRAS Q61H) were injected subcutaneously into SCID mice. Once tumors reached 5-10 mm in diameter, voruciclib or vehicle were administered orally at 50, 100, or 200 mg/kg OD for 11-14 days. Tumors were measured every 2-3 days, and growth inhibition relative to control was calculated. Significant tumor growth inhibition (>50%) was observed at all doses of voruciclib tested. Collectively, these data demonstrate that voruciclib inhibition of CDK9 leads to reduced phosphorylation of MYC on Ser 62 followed by a decrease in total MYC protein in MIA PACA-2 cells and inhibition of growth in multiple KRAS mutant cancer cell lines in vivo and in vitro. This suggests that voruciclib could be an attractive therapeutic option for cancers driven by KRAS-MYC.

Citation Format: Sandra E. Wiley, Yongwei Su, Yubin Ge. Voruciclib, a CDK9 inhibitor, downregulates MYC and inhibits proliferation of KRAS mutant cancers in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1962.
Abstract 2007: Investigating how RIPK1-mediated mitophagy induces cell death in ECM-detached cancer cells
Authors: Cockfield Jordan Alexandria , Antony Ishan , Ekanayake Samantha , Schafer Zachary T.
Published: July 2021

Lead Contact Metastasis is responsible for 90% of deaths caused by cancer. For cancer cells to begin successful metastasis, they must detach from their extracellular matrix (ECM) and evade intrinsic cell death mechanisms. Detachment from the ECM is known to induce a caspase-dependent cell death program, termed anoikis, that cancer cells have been shown to circumvent. Nonetheless, overcoming caspase-dependent cell death alone is not sufficient to promote survival during ECM-detachment, but cancer cells must also address caspase-independent cell death programs. In our recent studies, we have identified RIPK1 as a critical mediator of ECM-detachment induced caspase-independent cell death via its capacity to promote mitophagy and subsequent accumulation of mitochondrial ROS. More recently, we have begun to examine how ROS levels are elevated as a consequence of mitophagy and how these ROS ultimately promote cell death. During mitophagy, autophagosomes containing mitochondria fuse with lysosomes containing a plethora of hydrolases that could facilitate cell death. As such, we sought to examine the relationship between RIPK1-mediated mitophagy and lysosomal membrane permeabilization (LMP) Using immunoblotting, we found that not only does ECM-detachment promote the expression of the lysosomal protease cathepsin B, but also that this induction is dependent upon RIPK1 activity. Given that mitochondrial ROS (mitoROS) was shown to be a consequence of RIPK1-mediated mitophagy during ECM-detachment, we investigated if mitoROS were required for the observed changes in cathepsin B expression. Indeed, when treated with a mitochondrial ROS (mitoROS) scavenger, cathepsin B protein levels noticeably reduce. Taken together, this data suggests that RIPK1-driven mitoROS production is playing a role in regulating cathepsin B expression during ECM-detachment. Future studies will be aimed at determining whether these RIPK1-mediated alterations in cathepsin B are linked to the induction of caspase-independent cell death.

Citation Format: Jordan Alexandria Cockfield, Ishan Antony, Samantha Ekanayake, Zachary T. Schafer. Investigating how RIPK1-mediated mitophagy induces cell death in ECM-detached cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2007.
Abstract 2010: Pancreatic cancer stem cell exosomes drive a hierarchical intratumor communication network that fuels disease progression through hippo pathway inhibition
Authors: Ruivo Carolina F. , Bastos Nuno , Batista Ines , Adem Barbara , Duraes Cecilia , Melo Carlos A. , Campos-Laborie Francisco J. , Moutinho-Ribeiro Pedro , Mourão Barbara , Cravo Marília , Macedo Guilherme , Carneiro Fátima , Kouzarides Tony , Kalluri Raghu , Machado Jose C. , Melo Sonia
Published: July 2021

Intra-tumor heterogeneity represents a major challenge for cancer treatment. Resistance to therapy and tumorigenic ability varies greatly between distinct subpopulations of cancer cells. Intercellular communication is critical to establish a cooperative environment that maintains tumor heterogeneity and potentiates disease progression. However, how subpopulations of cancer cells interact to support tumor progression is not fully understood. Exosomes have the potential to re-educate recipient cells through the delivery of their cargo, emerging as the main mediators of intercellular communication. Here we demonstrate that subpopulations of pancreatic cancer cells use exosomes to establish a hierarchical and dynamic communication network, the ExoNet. Exosomes from cancer stem cells (CSC) are preferentially taken up by non-CSC (NCSC). Targeted inhibition of CSC exosomes in PDAC orthotopic, genetically engineered and patient-derived xenograft mouse models is sufficient to impair tumor growth and metastasis establishment. A comprehensive comparative proteomic analysis of exosomes derived from distinct subpopulations of pancreatic ductal adenocarcinoma (PDAC) human cell lines identified a significant enrichment of Agrin in CSC exosomes. Blocking exosomes secretion and downregulating Agrin in CSCs significantly diminishes YAP nuclear localization in receiving cells, indicating that CSC exosomes contribute for Hippo pathway inactivation. Agrin+ circulating exosomes correlate with tumor burden in PDAC patients, and most importantly correlate with CD133+ circulating exosomes, indicating that circulating Agrin+ exosomes are from CSC origin. Longitudinal analysis of circulating Agrin+ exosomes in PDAC patients predicts disease progression and response to therapy. Our study describes a novel role of cancer exosomes in the interplay between subpopulations of cancer cells promoting tumor progression. We identified Agrin as a potential non-invasive biomarker to monitor disease progression and predict treatment response.

Citation Format: Carolina F. Ruivo, Nuno Bastos, Ines Batista, Barbara Adem, Cecilia Duraes, Carlos A. Melo, Francisco J. Campos-Laborie, Pedro Moutinho-Ribeiro, Barbara Mourão, Marília Cravo, Guilherme Macedo, Fátima Carneiro, Tony Kouzarides, Raghu Kalluri, Jose C. Machado, Sonia Melo. Pancreatic cancer stem cell exosomes drive a hierarchical intratumor communication network that fuels disease progression through hippo pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2010.
Abstract 2027: SETD2 loss in renal carcinoma cells induces the unfolded protein response
Authors: Metz Alexander , Uzzo Robert , Abbosh Philip
Published: July 2021

Introduction: SETD2 encodes a histone H3-K36 methyltransferase which is frequently inactivated in clear cell renal carcinomas (ccRCCs) and papillary RCCs via 3p deletion/LOH and deleterious mutations. Histone H3-K36 trimethylation is facilitated by SETD2, which is necessary for proper pre-mRNA intron splicing. Improperly spliced mature-mRNA may lead to aberrant translation of retained introns (ATaRI), which represents potential therapeutic vulnerabilities. We explored this hypothesis using real world data and RCC models.

Methods and Results: Gene set enrichment analysis comparing SETD2-mutant to WT tumors using samples from the TCGA KIRC data set revealed that the untranslated protein response (UPR) was strongly enriched, as well as several immunotherapy-relevant pathways. This suggested that peptides arising from ATaRI may be present, since they would not be expected to fold properly and thus need to be addressed by the UPR pathway to maintain homeostasis. To investigate this further, we generated Setd2-isogenic RENCA cells using CRISPR. Knockout was confirmed by sequencing and immunoblot. H3K36 trimethylation was decreased or eliminated in monoclonal knockout cell lines, confirming a functional effect. Markers of UPR activation, including cleaved Atf6 and Atf4, were found to be upregulated in Setd2-mutant RENCA cells compared to controls as measured by immunoblot. Cleaved ATF6 is known to translocate to the nucleus to take part in a UPR transcriptional program. Consistent with this, ATF6 was found to localize to the nucleus in Setd2-knockout cells using immunofluorescence. Interestingly, CHOP, another downstream effector of the UPR pathway which predominantly regulates cell death, did not become upregulated in Setd2-knockout cells, suggesting activation of a compensatory cell survival pathway.

Conclusions: We identify activation of the UPR upon Setd2 loss and suggest that activation of part of the UPR pathway may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. We continue to evaluate the generation of peptides arising from ATaRI in Setd2-mutant contexts.

Citation Format: Alexander Metz, Robert Uzzo, Philip Abbosh. SETD2 loss in renal carcinoma cells induces the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2027.
Abstract 2039: The role of HORMAD1 in DNA damage repair in squamous cell carcinomas
Authors: Gantchev Jennifer , Villarreal Amelia Martinez , Ramchatesingh Brandon , Litvinov Ivan V.
Published: July 2021

Genomic instability is a prominent hallmark of cancer, however the dynamic mechanisms that drive and sustain genomic instability remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability express meiosis genes and undergo a process called meiomitosis. Cancer meiomitosis is the orchestrated activation of both mitotic and meiotic machineries in neoplastic cells that confer a selective advantage. Specifically, meiCT (meiosis-specific cancer/testis) genes that specialize in the reductional division of germ cells in meiosis I are ectopically expressed in several cancers, particularly in squamous cell carcinomas. The meiCT gene, HORMAD1 is a meiosis specific protein that functions to ensure that a sufficient number of DSBs are formed for appropriate meiotic progression and to maintain genome integrity throughout homologous recombination. Studies show that HORMAD1 is significantly upregulated in several cancers and is regarded as a potentially important oncogene that plays a role in sustaining increased genomic instability. With the use of shRNA mediated knockdown of HORMAD1, we evaluated the effects ofHORMAD1 on genomic instability and survival in squamous cell carcinomas (SCCs). Our results demonstrate that HORMAD1 knockdown in SCC cancer cell lines leads to an increase in genomic instability denoted by an increase in double strand breaks, micronuclei formation and ploidy. Furthermore, shHORMAD1 results in increased sensitivity to etoposide treatment leading to increased centrosome formation and reduced cell survival. We also demonstrate thatHORMAD1 modulates a functional and balanced level of genomic instability by eliciting an DNA damage response through homologous recombination mechanisms that allows SCCs to thrive following DNA damage.

Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in DNA damage repair in squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2039.
Abstract 204: Identification of gene expression signatures as potential novel biomarkers in malignant melanoma
Authors: Figueroa Stephanie , Tiwari Raj , Geliebter Jan
Published: July 2021

Melanoma accounts for the vast majority of skin cancer-related deaths due to its ability to metastasize and therapeutic resistance. We aimed to identify expression-based signatures associated with melanoma patient survival by analyzing The Cancer Genome Atlas (TCGA) database to identify potential, prognostic biomarkers of cutaneous melanoma. Genes overexpressed in melanoma, compared with normal skin, as well as genes associated with the survival of melanoma patients were found using the online bioinformatics platform GEPIA2 (Gene Expression Profiling Interactive Analysis). The Metascape bioinformatics tool was then used for clustering of the genes based on processes and Protein-Protein Interaction (PPI) Enrichment Analysis. The genes from the 6 most significantly enriched pathways were selected to create gene signatures, which were then subjected to Kaplan-Meier survival analysis. Melanoma patients from the TCGA database (458) were placed into two groups (229 patients each): a high and a low expression group. There were 139 statistically significantly upregulated genes in melanoma (p<0.01), while 500 genes had expression levels that were significantly associated with the survival of melanoma patients. Overexpressed genes were found to belong to pigment-related pathways (pigmentation, cellular pigmentation, and pigment cell differentiation), while survival-associated genes were found to function in immune system-related pathways (adaptive immune response, lymphocyte activation, and immune response-regulating signaling pathways). Kaplan-Meier survival analysis showed that the most overexpressed genes in melanoma correlated with poorer survival, while higher expression of the most-significantly survival associated gene signatures correlated with better survival. Together, these gene signatures may serve as predictors of survival in melanoma patients.

Citation Format: Stephanie Figueroa, Raj Tiwari, Jan Geliebter. Identification of gene expression signatures as potential novel biomarkers in malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 204.