Showing papers by "Robert H. Shoemaker published in 2022"

Clinical grade ACE2 as a universal agent to block SARS‐CoV‐2 variants

Abstract: The recent emergence of multiple SARS‐CoV‐2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS‐CoV‐2 variants. Here, we report that all SARS‐CoV‐2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS‐CoV‐2 isolates. Effective inhibition of infections with SARS‐CoV‐2 variants was validated and confirmed in two independent laboratories. These data show that SARS‐CoV‐2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan‐SARS‐CoV‐2 therapeutic.

Development of an aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer

Abstract: As ACE2 is the critical SARS-CoV-2 receptor, we hypothesized that aerosol administration of clinical grade soluble human recombinant ACE2 (APN01) will neutralize SARS-CoV-2 in the airways, limit spread of infection in the lung, and mitigate lung damage caused by deregulated signaling in the renin-angiotensin (RAS) and Kinin pathways. Here, after demonstrating in vitro neutralization of SARS-CoV-2 by APN01, and after obtaining preliminary evidence of its tolerability and preventive efficacy in a mouse model, we pursued development of an aerosol formulation. As a prerequisite to a clinical trial, we evaluated both virus binding activity and enzymatic activity for cleavage of Ang II following aerosolization. We report successful aerosolization for APN01, retaining viral binding as well as catalytic RAS activity. Dose range-finding and IND-enabling repeat-dose aerosol toxicology testing were conducted in dogs. Twice daily aerosol administration for two weeks at the maximum feasible concentration revealed no notable toxicities. Based on these results, a Phase I clinical trial in healthy volunteers has now been initiated (NCT05065645), with subsequent Phase II testing planned for individuals with SARS-CoV-2 infection.

Targeting Glutamine Metabolism to Enhance Immunoprevention of EGFR‐Driven Lung Cancer

Abstract: Lung cancer is the leading cause of cancer death worldwide. Vaccination against EGFR can be one of the venues to prevent lung cancer. Blocking glutamine metabolism has been shown to improve anticancer immunity. Here, the authors report that JHU083, an orally active glutamine antagonist prodrug designed to be preferentially activated in the tumor microenvironment, has potent anticancer effects on EGFR‐driven mouse lung tumorigenesis. Lung tumor development is significantly suppressed when treatment with JHU083 is combined with an EGFR peptide vaccine (EVax) than either single treatment. Flow cytometry and single‐cell RNA sequencing of the lung tumors reveal that JHU083 increases CD8+ T cell and CD4+ Th1 cell infiltration, while EVax elicits robust Th1 cell‐mediated immune responses and protects mice against EGFRL858R mutation‐driven lung tumorigenesis. JHU083 treatment decreases immune suppressive cells, including both monocytic‐ and granulocytic‐myeloid‐derived suppressor cells, regulatory T cells, and pro‐tumor CD4+ Th17 cells in mouse models. Interestingly, Th1 cells are found to robustly upregulate oxidative metabolism and adopt a highly activated and memory‐like phenotype upon glutamine inhibition. These results suggest that JHU083 is highly effective against EGFR‐driven lung tumorigenesis and promotes an adaptive T cell‐mediated tumor‐specific immune response that enhances the efficacy of EVax.

Methylated Septin9 (mSEPT9): A Promising Blood-Based Biomarker for the Detection and Screening of Early-Onset Colorectal Cancer

Abstract: Early-onset colorectal cancer (EOCRC), defined as a diagnosis under age 50, is an emerging public health burden. As many of these individuals fall outside of screening guidelines, the development of a minimally invasive, accurate screening modality for this population is warranted. We evaluated the FDA-approved blood-based biomarker methylated Septin9 (mSEPT9) test as screening tool for EOCRC. EOCRC plasma, healthy plasma, and serum-free conditioned media from cancer cell lines were collected. Cell-free DNA (cfDNA) was isolated and bisulfite converted for use in the assay. mSEPT9 and ACTB measured using Epi proColon V2.0. EOCRC plasma was collected at Massachusetts General Hospital (2005–2019) and controls were collected at the NIH and by ZenBio Inc. (prior to 2019). Twenty-seven EOCRC cases, 48 healthy controls <50 years old, and 39 healthy controls ≥50 years old were included in this study. mSEPT9 was detected more frequently in EOCRC cases (88.9%) compared with healthy controls age <50 (4.2%) and ≥50 (15.4%), respectively (P < 0.001). The sensitivity, specificity, positive predictive value, and negative predictive values of the mSEPT9 assay to detect EOCRC was 90.8% (95% CI, 84.7%–96.9%), 88.9% (95% CI, 77.0%–100.0%), 96.3% (95% CI, 92.3%–100.0%), and 75.0% (95% CI, 60.0%–90.0%), respectively, compared with all healthy controls. mSEPT9 cfDNA level was an independent predictor of survival (P = 0.02). mSEPT9 is a sensitive and specific biomarker for EOCRC detection. These results suggest that mSEPT9 may be useful in the detection of EOCRC, providing a minimally invasive method for screening in this growing population of patients with colorectal cancer. Significance: mSEPT9 may be a novel biomarker for the detection of early-onset colorectal cancer, as it demonstrated high sensitivity and specificity in our study.

Mesothelioma Mouse Models with Mixed Genomic States of Chromosome and Microsatellite Instability

Abstract: Simple Summary Only a limited number of murine mesothelioma cell lines have been developed to date. We sought to expand this number and to characterize the models in detail to enable studying mesothelioma biology in vivo. Two cell lines were identified as showing well-defined mesothelioma biomarkers and being suitable for preclinical use. In the course of our studies, we observed a mixed phenotype of chromosomal instability and microsatellite instability not previously reported in mouse models. Moreover, microsatellite markers were detectable in the plasma of tumor-bearing animals, which potentially can be used as non-invasive biomarkers for early cancer detection and monitoring the effects of interventions. Abstract Malignant mesothelioma (MMe) is a rare malignancy originating from the linings of the pleural, peritoneal and pericardial cavities. The best-defined risk factor is exposure to carcinogenic mineral fibers (e.g., asbestos). Genomic studies have revealed that the most frequent genetic lesions in human MMe are mutations in tumor suppressor genes. Several genetically engineered mouse models have been generated by introducing the same genetic lesions found in human MMe. However, most of these models require specialized breeding facilities and long-term exposure of mice to asbestos for MMe development. Thus, an alternative model with high tumor penetrance without asbestos is urgently needed. We characterized an orthotopic model using MMe cells derived from Cdkn2a+/−;Nf2+/− mice chronically injected with asbestos. These MMe cells were tumorigenic upon intraperitoneal injection. Moreover, MMe cells showed mixed chromosome and microsatellite instability, supporting the notion that genomic instability is relevant in MMe pathogenesis. In addition, microsatellite markers were detectable in the plasma of tumor-bearing mice, indicating a potential use for early cancer detection and monitoring the effects of interventions. This orthotopic model with rapid development of MMe without asbestos exposure represents genomic instability and specific molecular targets for therapeutic or preventive interventions to enable preclinical proof of concept for the intervention in an immunocompetent setting.

Oral administration of TRAIL-inducing small molecule ONC201/TIC10 prevents intestinal polyposis in the Apc min/+ mouse model.

Abstract: Colorectal cancer (CRC) incidence is rising globally. Hence, preventing this disease is a high priority. With this aim, we determined the CRC prevention potential of the TRAIL-inducing small molecule ONC201/TIC10 using a preclinical model representing high-risk familial adenomatous polyposis (FAP) patients, Apc min/+ mice. Prior to the efficacy study, optimal and non-toxic doses of ONC201 were determined by testing five different doses of ONC201 (0-100 mg/kg body weight (BW); twice weekly by oral gavage) in C57BL/6J mice (n=6/group) for 6 weeks. BW gain, organ weights and histopathology, blood profiling, and the plasma liver enzyme profile suggested no toxicities of ONC201 at doses up to 100 mg/kg BW. For efficacy determination, beginning at six weeks of age, groups of Apc min/+ male and female mice (n≥20) treated with colon carcinogen azoxymethane (AOM) (AOM-Apc min/+) were administered ONC201 (0, 25, and 50 mg/kg BW) as above up to 20 weeks of age. At termination, efficacy was determined by comparing the incidence and multiplicity of intestinal tumors between vehicle- and drug-treated groups. ONC201 showed a strong suppressive effect against the development of both large and small intestinal tumors in male and female mice. Apc min/+ mice treated with ONC201 (50 mg/kg BW) showed >50% less colonic tumor incidence (P<0.0002) than controls. Colonic tumor multiplicity was also significantly reduced by 68% in male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in controls; P<0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in controls; P<0.0003) with ONC201 treatment (50 mg/kg BW). Small intestinal polyps were reduced by 68% in male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in controls; P<0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in controls; P<0.0001). Molecular analysis of the tumors suggested an increase in TRAIL, DR5, cleaved caspases 3/7/8, Fas-associated death domain protein (FADD), and p21 (WAF1) in response to drug treatment. Serum analysis indicated a decrease in pro-inflammatory serum biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated mice compared with controls. Our data demonstrated excellent chemopreventive potential of orally administered ONC201 against intestinal tumorigenesis in the AOM-Apc min/+ mouse model.

Characterization of stage‐specific tumor progression in TMPRSS2‐ERG (fusion)‐driven and non‐fusion‐driven prostate cancer in GEM models

Abstract: In the present study, we performed a comparative stage‐specific pathological and molecular marker evaluation of TMPRSS2‐ERG fusion and PTEN loss‐driven (TMPRSS2‐ERG. Ptenflox/flox) versus non‐fusion‐driven prostate tumorigenesis (Hi‐Myc) in mice. Anterior, ventral, and dorsolateral prostates were collected from mice at different ages (or time points post‐Cre induction). Results indicated that growth and progression of prostatic intraepithelial lesions to adenocarcinoma stages occurred in both mice models albeit at different rates. In the TMPRSS2‐ERG. Ptenflox/flox mice, the initiation of tumorigenesis was slow, but subsequent progression through different stages became increasingly faster. Adenocarcinoma stage was reached early on; however, no high‐grade undifferentiated tumors were observed. Conversely, in the Hi‐Myc+/− mice, tumorigenesis initiation was rapid; however, progression through different stages was relatively slower and it took a while to reach the more aggressive phenotype stage. Nevertheless, at the advanced stages in the Hi‐Myc+/− mice, high‐grade undifferentiated tumors were observed compared to the later stage tumors observed in the fusion‐driven TMPRSS2‐ERG. Ptenflox/flox mice. These results were corroborated by the stage specific‐pattern in the molecular expression of proliferation markers (PCNA and c‐Myc); androgen receptor (AR); fusion‐resultant overexpression of ERG; Prostein (SLC45‐A3); and angiogenesis marker (CD‐31). Importantly, there was a significant increase in immune cell infiltrations, which increased with the stage of tumorigenesis, in the TMPRSS2‐ERG fusion‐positive tumors relative to fusion negative tumors. Together, these findings are both novel and highly significant in establishing a working preclinical model for evaluating the efficacy of interventions during different stages of tumorigenesis in TMPRSS2‐ERG fusion‐driven PCa.

Abstract A018: Targeting STAT3 for bladder cancer prevention – in vitro studies using spheroid and organoid models

Abstract: Signal Transducer and Activator of Transcription 3 (STAT3) is tightly regulated in normal cells to maintain a transiently active state. In contrast, persistent STAT3 activation is frequently observed in bladder cancer (BC) and is associated with poor prognosis and chemoresistance. Hence, developing small molecule inhibitors targeting STAT3 may be helpful for preventing BC progression and improving the survival rate of patients with metastatic BC. Recently, the use of three-dimensional in vitro models in drug development has gained popularity as they closely resemble, to an extent, the in vivo environment in heterogeneity and physiological conditions. Here we established spheroid and organoid models for bladder cancer and evaluated STAT3 inhibitors (C188-9 and SH5-07) for their anticancer activity in vitro. Initially, we optimized the spheroid growth from human, rat, and mouse BC cell lines (J82, NBT-II, MB49, respectively) and tumoroid growth from the BBN-rat bladder cancer model. The anticancer efficacy of C188-9 and SH5-07 was evaluated in vitro at various doses (0-50 µM) in the 3D models of BC. Assays were performed to determine spheroid viability (calcein AM (CA) and EtBr staining), ATP and ROS production (MitoSOX™). Protein isolated from control and drug treated spheroids/tumoroids was used to evaluate pharmacodynamic biomarkers of cell proliferation, apoptosis, and STAT3 signaling. We demonstrate that treatment with C188-9 and SH5-07 significantly decreased the spheroids size (39-45% smaller compared to untreated, p<0.0001) along with decreased ATP (20%-40%, p<0.05), and pSTAT3 protein expression in spheroids derived from BC cell lines and rat BC organoids. Further, MitoSOX™ staining showed that STAT3 inhibitor treatment induced mitochondrial mediated ROS generation in BC spheroids. CA and EtBr staining showed that C188-9 and SH5-07 treatment induced cell death in BC spheroids that was also associated with caspase-3 cleavage. These findings indicate that C188-9 and SH5-07 could suppress the activation of the STAT3 pathway and inhibit the bladder cancer spheroid growth by inducing ROS production and thus warrants further evaluation in vivo. Furthermore, our study provided valuable spheroid and organoid models for evaluating therapeutic candidates in an in vivo-mimic microenvironment, thereby providing great potential for drug testing. (Partly supported by P30CA225520 and Kerley-Cade Endowed Chair) Citation Format: Surya P Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Targeting STAT3 for bladder cancer prevention – in vitro studies using spheroid and organoid models [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A018.

Abstract IA015: Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination

Abstract: Top2A is a key enzyme involved in DNA replication and is a therapeutic target for several cancer types including breast cancer. Overexpression of Top2A has been observed in both human and mouse triple-negative breast cancer (TNBC). The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multi-peptide vaccine targeting multiple epitopes of the Top2A protein. Top2A-specific MHC II epitopes with optimal binding affinity were identified using a combined scoring system, which predicted their potential to elicit a Th1 immune response. The formulated vaccine contained top three Top2A peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. Anti-tumor efficacy of the Top2A vaccine was initially evaluated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to the adjuvant controls. The Top2A peptide vaccine exhibited striking efficacy in a genetically engineered TNBC mouse model (C3(1)/Tag), reducing tumor burden by >90% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. There were no overt toxicities observed with the Top2A vaccination. To explore potential mechanisms underlying the anti-tumor response induced by Top2A vaccine treatment, scTCR-seq of tumors in both control and Top2A vaccine groups revealed new T cell clones as a consequence of Top2A vaccination. Furthermore, in vitro stimulation of these splenocytes by the vaccinated Top2A peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. Our data indicate that the newly developed multi-peptide Top2A vaccine is immunogenic and efficacious in the prevention of TNBC development and progression in vivo. Citation Format: Sang Beom Lee, Jing Pan, Donghai Xiong, Katie Palen, Bryon Johnson, Jeffrey E. Green, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA015.

Abstract 5562: Development of a TERT-specific peptide/adjuvant vaccine in C57BL/6 mice

Abstract: Telomerase plays a vital role in cell senescence and cellular replication and has been described as a leading regulator in several human cancers, including prostate, thyroid, breast, colon, and bladder cancer. Telomerase is responsible for elongating telomere length and prolonging cellular lifespan as well as acting as a transcriptional modulator in cancer signaling pathways. Because of its enhanced expression in primary tumor cells as well as incipient tumor-initiating stem cells and with limited distribution in normal somatic cells, the catalytic component of telomerase, TERT (telomerase reverse transcriptase), is regarded as a high-impact target for immunotherapeutic agents and vaccines. In order to derive a mouse TERT (mTERT)-specific vaccine which could be useful in a preventive approach, we implemented an epitope-mapping stratagem by which pools of 30-mer peptides overlapping by 5 spanning the entire mTERT sequence were used to immunize C57BL/6 mice. Several immunogenic peptides were identified by matrixed peptide pool IFN-ɣ ELISPOT screening in a 2-part process that first identified positive pools, then identified individual peptides. 15-mer and 31-mer versions of the top 7 immunogenic mTERT peptides were synthesized and used as a peptide pool combined separately with several adjuvants to optimize immunogenicity. Results suggested a peptide sequence-specific preference for TLR3-mediated Hiltonol versus TLR9-mediated CpG-based adjuvants. In addition, the majority of T cell responses specific to the TERT peptides was identified through flow cytometry as belonging to the CD4 compartment, although one class I-restricted epitope was also identified. Multifunctional CD4+ T cells expressing IFN-ɣ, TNF-α, IL-2, and CD107a were identified specific to several mTERT peptides. In vivo cytotoxic effects exerted on TERT peptide pool-loaded target cells were also uncovered that were specific to a CpG-adjuvanted version of the vaccine. Finally, multiple versions of the mTERT peptide/adjuvant vaccine with highest immunogenicity scores are being tested in a syngeneic graft mouse model using subcutaneous implantation of lung cancer-derived SPON10 cells expressing mTERT. If proven efficacious, these results will support the feasibility of a TERT peptide/adjuvant approach for prophylactically engendering broad antitumor immunity to TERT-expressing cancers. Funded by NCI Contract No. HHSN261200800001E Citation Format: Jason D. Marshall, Yurong Song, Hamid Zarkesh, Rebecca L. Matthews, Chelsea Sanders, Simone Difilippantonio, Ligia A. Pinto, Shizuko Sei, Robert H. Shoemaker. Development of a TERT-specific peptide/adjuvant vaccine in C57BL/6 mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5562.

Targeting the Leukotriene Pathway for Colon Cancer Interception.

Abstract: The role of chronic inflammation and arachidonic acid (AA) metabolism in tumor progression has been well characterized for variety of cancers, with compelling data for colon cancer. Several preclinical and clinical studies primarily focused on inhibiting the cyclooxygenase pathways using NSAIDs and aspirin for colon cancer prevention. However, emerging evidence clearly supports the pro-tumorigenic role of 5-lipoxygenase and its downstream leukotriene pathway within AA metabolism. As discussed in the current issue, targeting the leukotriene pathway by cysteinyl leukotriene receptor antagonist (LTRA) montelukast suppressed formation of aberrant crypt foci (ACF) and cell proliferation in colonic epithelium, suggesting the potential of LTRAs for colon cancer prevention. Although this is a short clinical chemoprevention trial to explore the effects of LTRAs against ACF development, it is a significant and timely study opening avenues to further explore the possibilities of using LTRAs in other inflammation-associated precancerous lesions as well. In this spotlight commentary, we highlight the implications of their data and the opportunities for developing LTRAs as potential candidates for colorectal cancer interception. See related article by Higurashi et al., p. 661.

Abstract IA007: PREVENT agent development pipeline

Abstract: The NCI’s PREVENT Cancer Preclinical Drug Development Program is a peer-reviewed program designed to support the preclinical development of promising agents and biomarkers for cancer interception/prevention towards clinical applications. PREVENT is not a grant program but allocates NCI contract resources to advance approved projects in a milestone-driven manner. Results obtained through NCI contract resources are returned to the applicant PIs and used to support further development by the applicants or in partnership with NCI. Resources available to PREVENT Program applicants include preclinical efficacy testing, CGMP manufacturing, GLP pharmacokinetic and IND-enabling toxicology studies, and IND filings. The PREVENT Program is focused on preventive agent development in the areas of Immunoprevention (cancer vaccines and immunomodulatory agents), Chemoprevention (novel mechanisms, anti-inflammatory agents, drug repurposing, toxicity reduction via alternative dosing regimens and agent combinations) and clinically translatable mechanistic biomarkers (pharmacodynamics, immune correlates, and tumor preventive efficacy). Submission deadlines for PREVENT Concept Applications occur twice per year on the second Monday in January and July. Further information can be obtained at the PREVENT Program website: https://prevention.cancer.gov/major-programs/prevent-cancer-preclinical Citation Format: Mark Stevn Miller, Brian Cholewa, John Clifford, Vignesh Gunasekharan, Altaf Mohammed, Shanker Gupta, Robert Shoemaker, Shizuko Sei. PREVENT agent development pipeline [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA007.