Acute graft-versus-host disease (GVHD) is a donor T cell driven complication and the leading cause of non-relapse mortality in patients receiving an allogeneic hematopoietic cell transplantation (allo-HCT). Allogeneic donor T cells eradicate residual leukemia and prevent relapse via the graft-versus-leukemia (GVL) effect and are critical for responding against opportunistic infections post-transplant. Current regimens successful in preventing GVHD are broadly immunosuppressive and come at the cost of increased risk of relapse and/or infection. Therefore, there is an urgent need for new approaches that limit GVHD while retaining GVL responses. During GVHD, alloreactive T cells boost their energy production through oxidative phosphorylation (OXPHOS) and glycolysis, supporting heightened proliferation and pathogenicity against healthy host tissues. The enzyme dihydroorate dehydrogenase (DHODH), is essential for de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential during OXPHOS. Having shown upregulation of DHODH messenger RNA and protein expression in activated human T cells, we evaluated DHODH inhibition, via a small molecule inhibitor HOSU-53, as a therapeutic approach for GVHD. Inhibiting DHODH significantly reduced oxidative metabolism in T cells both during and after activation, while selectively suppressing inflammatory cytokine production in de novo activated, but not previously activated, T cells. In a xenogeneic model, HOSU-53 treatment limited GVHD severity, decreased pathogenic Th1 and Th17 response, and preserved beneficial GVL effects. Altogether, we identify DHODH inhibition as an innovative treatment strategy in allo-HCT recipients to reduce GVHD severity and retain effective GVL response.

Objectives: In rapidly proliferating cancer cells, the de novo pyrimidine synthesis pathway is highly activated and enhances the tumor’s supply of pyrimidine nucleotides. HOSU-53 is under development as an orally bioavailable, small-molecule inhibitor of dihydroorotate dehydrogenase (DHODH), a mitochondrial enzyme that catalyzes the rate-limiting step of de novo pyrimidine nucleotide biosynthesis, conversion of dihydroorotate (DHO) to orotate1. Biological testing verified the efficacy of HOSU-53 in acute myeloid leukemia, multiple myeloma, small-cell lung cancer, melanoma, and other cancer cell lines. Our goals were to develop PK/PD models to scale across species and utilize plasma DHO exposure as a biomarker for both efficacy and on-target toxicity to target a safe, yet potentially effective starting dose in a first-in-human (FIH) trial.Methods: Plasma HOSU-53 and DHO concentration vs. time data were available from Good Laboratory Practice (GLP) and non-GLP pharmacokinetics (PK), toxicity, and efficacy studies in mice, rats, and beagle dogs. One-, two-, and three-compartment models for HOSU-53 PK were investigated and then linked to various PD models for DHO accumulation via DHODH inhibition, and a comprehensive dataset combining pharmacokinetics/pharmacodynamics (PK/PD) data across the three species was used for fitting and estimation of random and fixed effects, including allometric scale factors. Simulations of various dose regimens in humans have been made based on the final PK/PD model selected.Results: A 2-compartment model with first-order absorption and linear elimination with allometric factors adequately characterized the observed HOSU-53 PK profiles across species. DHO response to HOSU-53 was best characterized using a turnover model to capture the delay between plasma drug concentration and biomarker response. Compared with a previously developed physiologically based PK (PBPK) model, the PK/PD model is in good agreement in predicting human PK profiles. However, the HOSU-53 exposure-DHO response relationships did not scale well across species, suggesting yet unidentified mechanistic components may be required within the model.Conclusions: A translational modeling and simulation approach was used to identify a target dose range for the FIH study of a novel DHODH inhibitor using data from preclinical studies. Human simulations suggest an oral FIH starting dose of 5 mg QD will be safe and near the low end of the predicted efficacy range based on preclinical DHO exposure data. The discrepancy in PD predictions between species indicates better understanding of the pharmacological mechanisms of HOSU-53, DHODH, de novo pyrimidine synthesis, and DHO pharmacodynamics across species is needed to aid the development of HOSU-53 in humans.Citations: [1] Elgamal, O. A.; Fobare, S.; Vibhute, S.; Mehmood, A.; Vroom, D. C.; Johnson, M. L.; Stearns, B.; Lerma, J. R.; Truxall, J.; Stahl, E.; Carmichael, B.; Orwick, S. J.; Mims, A. S.; Curran, E.; Santhanam, R.; Tridandapani, S.; Phelps, M. A.; Xie, Z.; Coss, C. C.; Baker, S. D.; Patrick, J.; Ezzell, J. K.; Rai, J.; Pan, J.; Rai, S. N.; Stillwell, C.; Wunderlich, M.; Abdulrahim, M.; Goodwin, T. E.; Hilinski, G.; Bennett, C. E.; Hertlein, E.; Byrd, J. C. Pyrimidine Depletion Enhances Targeted and Immune Therapy Combinations in Acute Myeloid Leukemia. JCI Insight 2024, 9 (8). https://doi.org/10.1172/jci.insight.173646.

Pyrimidine biosynthesis, as a precursor of RNA and DNA, is essential for cell proliferation. Targeting pyrimidine metabolism with chemotherapy has been a treatment backbone for many cancers. Dihydroorotate dehydrogenase (DHODH) is an enzyme that is critical for de novo pyrimidine biosynthesis. Our team designed and synthesized an orally bioavailable small-molecule DHODH inhibitor, JBZ-001 (HOSU-53), as a candidate with good drug-like properties and oral bioavailability in mice and rats. In vivo testing of JBZ-001 established efficacy in multiple mouse xenograft models: small-cell lung cancer (NCI-H82 xenograft, tumor growth inhibition, TGI = 84%), colorectal cancer (HCT-15 xenograft, TGI = 91%), lymphoma (Z-138 xenograft, TGI = 102%), gastric cancer (SNU-16 xenograft, TGI = 88%), and melanoma (A375 xenograft, TGI = 64%) (Bennett C, ACS 2024). It showed superior preclinical efficacy when compared with other clinical candidates. It also showed a favorable toxicity profile with no observed adverse effects levels identified in the preclinical toxicity studies in rats and dogs. JBZ-001 has been moved to phase 1 testing in humans. The JBZ-001- phase 1 study will characterize the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of JBZ-001 as a single agent in patients with advanced solid tumors and non-Hodgkin lymphoma (NHL). Dose escalation will identify the optimal biological dose (Part 1), and the dose expansion (Part 2). We will test preliminary activity in cohorts of up to 10 patients with the same tumor type. A Bayesian safety monitoring rule will be used to evaluate the rate of dose-limiting toxicities (DLTs). Eligible patients are adults ≥18 years of age with confirmed relapsed or refractory advanced solid tumors and NHL, for which no standard approved treatment is available; have measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1; and an ECOG performance status of 0-2. The primary endpoint is determination of the optimal dose by assessment of specific DLTs and adverse events per Common Terminology Criteria for Adverse Events (CTCAE) v.5. Secondary endpoints include PK and PD parameters, objective response rate per RECIST v1.1, and duration of response. Safety and antitumor activity endpoints will be summarized using descriptive statistics. Recruitment is ongoing for Part 1 at The Ohio State University Comprehensive Cancer Center (WCG IRB 20245148). Asrar Alahmadi, Chad Bennett, Sebastian Biglione, Zuzana, Jirakova, Carly JR Pilcher, Ridge Archer, Tamara Jovonovich, Dwight Owen, Christian Rolfo, Robert Wesolowski, Claire Verschraegen, David Carbone. An open-label phase 1 study to investigate JBZ001 in adults with advanced solid tumors and non-Hodjkin lymphoma (JBZ001, trial in progress) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT199.

Small-cell lung cancer is an aggressive subtype of lung cancer with poor prognosis and poor overall survival and comprises approximately 15% of all lung cancers. SCLC, unlike NSCLC, has no known targetable driver mutations and, therefore, targeted therapeutics have been lacking. The frontline therapy for SCLC is platinum-etoposide chemotherapy, a regimen that has been in use for 30 years. Although patients respond well to this combination initially, they almost always relapse shortly after the start of therapy and second line therapies typically provide only a few months of benefit. Clearly, better therapeutics are necessary for the treatment of SCLC. Cancer cells often have dysregulated metabolic pathways. These changes are often necessary to enable the continued proliferation of the cancer cells. Therefore, key metabolic enzymes can have a crucial role in tumor cells survival and can be considered targets for cancer treatment. According to some recent studies, dihydroorotate dehydrogenase (DHODH), which catalyzes the conversion of dihydroorotate to orotate in the pyrimidine de novo synthesis pathway, has a particular role in the survival of different types of cancers including leukemia and SCLC. We evaluated the effect of HOSU-53, a novel DHODH inhibitor, on SCLC tumor growth in vitro and in vivo. We measured IC50s of 18 SCLC cell lines in vitro using CellTiter-Glo® Luminescent Cell Viability Assay and most SCLC cells showed sensitivity to HOSU-53 treatment in the low nanomolar range. Additionally, to determine the role of salvage pathway in HOSU-53 sensitivity, we treated the sensitive and resistant cells in parallel with HOSU-53 alone or in combination with exogenous uridine. In the presence of uridine, cell viability was significantly rescued in resistant cells compared to HOSU-53 treatment as single agent. However, we did not observe rescued cell viability in sensitive SCLC cells. These results suggesting that in SCLC cells which are resistant to de novo pyrimidine inhibition, activation of salvage pathway may be one of the compensatory mechanisms for tumor cells to sustain their survival. To evaluate the effect of DHODH inhibition on SCLC tumor growth in vivo, we treated xenograft mice models with HOSU-53 alone or in combination with etoposide plus cisplatin as the standard chemotherapy for SCLC. We observed a significant decrease of tumor volume when HOSU-53 was used alone and in combination, without significant animal weight loss. Together, these data demonstrated the promising efficacy of HOSU-53 in different SCLC subtypes and support the strategy of targeting DHODH as a potential therapy to treat SCLC. These findings support the initiation of a phase l clinical trial to evaluate the preliminary efficacy and tolerability of HOSU-53; therefore, we have initiated a first-in-human phase I trial of this agent. Bahareh Nourmohammadi, Ola A. Elgamal, Sandip Vibhute, Christopher C. Coss, Thomas E. Goodwin, Erin Hertlein, Joseph M. Amann, Chad E. Bennett, John C. Byrd, David P. Carbone. Promising therapeutic effects of pyrimidine synthesis inhibition by a novel dihydroorotate dehydrogenase inhibitor in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6902.

SIGNIFICANCE:Characterisation of tobacco product emissions is an important step in assessing their impact on public health. Accurate and repeatable emissions data require that a leak-tight seal be made between the smoking or vaping machine and the mouth-end of the tobacco product being tested. This requirement is challenging because of the variety of tobacco product mouth-end geometries being puffed on by consumers today. We developed and tested a prototype universal smoking machine adaptor (USMA) that interfaces with existing machines and reliably seals with a variety of tobacco product masses and geometries. METHODS:Emissions were machine-generated using the USMA and other available adaptors for a variety of electronic cigarettes (n=7 brands), cigars (n=4), cigarillos (n=2), a heated tobacco product, and a reference cigarette (1R6F), and mainstream total particulate matter (TPM) and nicotine were quantified. Data variability (precision, n≥10 replicates/brand) for all products and error (accuracy) from certified values (1R6F) were compared across adaptors. RESULTS:TPM and nicotine emissions generated using the USMA were accurate, precise and agreed with certified values for the 1R6F reference cigarette. Replicate data indicate that USMA repeatability across all tobacco products tested generally meets or exceeds that from the comparison adaptors and extant data. CONCLUSION:The USMA seals well with a variety of combustible tobacco products, e-cigarettes with differing geometries and plastic-tipped cigarillos. Variability for all measures was similar or smaller for the USMA compared with other adaptors.

Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.

We used a structure-based drug discovery approach to identify novel inhibitors of human dihydroorotate dehydrogenase (DHODH), which is a therapeutic target for treating cancer and autoimmune and inflammatory diseases. In the case of acute myeloid leukemia, no previously discovered DHODH inhibitors have yet succeeded in this clinical application. Thus, there remains a strong need for new inhibitors that could be used as alternatives to the current standard-of-care. Our goal was to identify novel inhibitors of DHODH. We implemented prefiltering steps to omit PAINS and Lipinski violators at the earliest stages of this project. This enriched compounds in the data set that had a higher potential of favorable oral druggability. Guided by Glide SP docking scores, we found 20 structurally unique compounds from the ChemBridge EXPRESS-pick library that inhibited DHODH with IC50, DHODH values between 91 nM and 2.7 mu M. Ten of these compounds reduced MOLM-13 cell viability with IC50, MOLM-13 values between 2.3 and 50.6 mu M. Compound 16 (IC50, DHODH = 91 nM) inhibited DHODH more potently than the known DHODH inhibitor, teriflunomide (IC50, DHODH = 130 nM), during biochemical characterizations and presented a promising scaffold for future hit-to-lead optimization efforts. Compound 17 (IC50, MOLM-13 = 2.3 mu M) was most successful at reducing survival in MOLM-13 cell lines compared with our other hits. The discovered compounds represent excellent starting points for the development and optimization of novel DHODH inhibitors.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Abstract Disclosure: P. Kumar: None. T.H. Helms: None. H. Radomska: None. W. Tyler: None. S.K. Kulp: None. C. Bennett: None. C.C. Coss: None. Background: Non-alcoholic fatty liver disease (NAFLD) is a major cause of chronic liver disease worldwide. NAFLD includes a range of conditions from simple steatosis to non-alcoholic steatohepatitis (NASH), advanced fibrosis, and cirrhosis that can ultimately lead to liver failure and/or hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) are essential to this pathologic fibrogenesis. In response to liver injury, they deposit collagen, fibronectin, and release the potent pro-fibrotic cytokine transforming growth factor-β (TGFβ). Estrogens have been shown to suppress the TGF-β-induced expression but the molecular mechanism is not completely understood. Estrogens are protective against multiple aspects of NAFLD/NASH pathophysiology. Their therapeutic potential however is limited by undesirable side effects attributed to ERα activation. Selective modulation of ERβ can provide therapeutic benefits of estrogens with reduced ERα-associated toxicities and has shown promising results in several preclinical NASH models. We hypothesize that beneficial estrogen pharmacology in NAFLD-NASH is driven by ERβ activation and ERβ-selective modulators can provide anti-NASH benefit. Our objective was to investigate the role of ERβ signaling and the therapeutic potential of the novel carborane ERβ-selective agonist, OSU-ERβ-12 (ERβ-12), in fibrosis and NASH models. Methods. Efficacy of ERβ-12 was tested in the CCl4 model of hepatic fibrosis by administering CCl4 to mice over 4 weeks at which point intervention with estradiol, LY500307 (LY) and ERβ-12 started. Combined treatment and CCL4 continued for 2 weeks when the CCl4 ceased, and therapy continued for 2 more weeks until sacrifice. We assessed fibrosis via algorithmic quantification of picrosirius red stain. Anti-fibrotic activity of ERβ-12 was assessed in vitro in a human HCC cell (HepG2)/human HSC (LX-2) co-culture system treated with ERβ-12, TGFβ or the combination for 24 h. Expression of pro-fibrotic, TGFβ pathway-related markers was assessed by qRT-PCR and western blotting in liver tissue and cell lysates. Results. ERβ-12 significantly reduced CCl4-induced liver fibrosis in mice at both non-selective (100 mg/kg) and ERβ-selective (10 mg/kg) doses. ERα-related toxicity (reduced urogenital weight) was not observed at the ERβ-selective dose. Reduced fibrosis was associated with decreased mRNA expression of pro-fibrotic and TGFβ pathway genes in livers of ERβ-12 treated mice. ERβ mRNA expression in HepG2/LX-2 co-culture cells was elevated compared to individual cell types. ERβ agonists (ERβ-12, LY) reduced TGFβ-mediated expression of pro-fibrotic genes: MCP-1, TGFβ, COL1A1. This effect was reversed with the pan-ER antagonist fulvestrant. Conclusion. These data suggest that ERβ agonism inhibits TGFβ-mediated liver fibrosis and supports further investigation of ERβ-targeted therapeutic candidates. Presentation: Saturday, June 17, 2023

Worldwide, multiple myeloma (MM) is the second most common hematological malignancy characterized by the expansion of aberrant mature plasma cells in the bone marrow. Despite the FDA approval of several drugs with different mechanisms of action such as immunomodulatory drugs (IMiDs) and proteosome inhibitors (PIs), MM remains a challenging incurable disease where most patients ultimately relapse and become refractory to available . The resistant nature of MM underscores the need for novel therapeutic strategies that can directly affect MM cells while enhancing the outcome with MM current standard of care (SOC). Among the most promising MM SOC agents is monoclonal antibodies (mAb) such as the CD38 antibody daratumumab, which significantly improved the management of newly diagnosed and relapsed/refractory MM. Therefore, we aimed to develop novel therapies that can enhance mAb therapy in MM. Dihydroorotate dehydrogenase (DHODH) is an enzyme which mediates the fourth and rate-limiting step in the de novo pyrimidine synthesis pathway converting dihydroorotate to orotate, the precursor of uridine. Pyrimidine starvation using DHODH inhibitors has been shown to have potent antiproliferative activity in several malignancies including acute myeloid leukemia (AML) and MM. Thus, we developed a novel potent DHODH inhibitor, HOSU-53 for the treatment of hematological malignancies. We previously discovered the ability of HOSU-53 to modulate surface CD38 expression and found that HOSU-53 had potent monotherapy activity in MM and provided impressive synergy in combination with daratumumab using the NCI-H929 MM cell line derived xenograft (CDX) model (2022 AACR annual meeting abstracts). Herein, we further expanded our studies to pre-clinically develop HOSU-53 as a new potential MM therapy and explore additional synergistic immunotherapy combinations that could maintain efficacy in the case of anti-CD38 therapy resistance. We evaluated the in vitro antiproliferative efficacy of HOSU-53 against a panel of MM cell lines and found nanomolar potency validating that HOSU-53 would have monotherapy efficacy. Indeed, we conducted in vivo studies using two additional MM CDX subcutaneous (s.c) models, OPM-2 and RPMI-8226, and found significant tumor delay and survival advantage. In the OPM-2 model, HOSU-53 has a median survival of 54-days compared to vehicle at 28-days, while the median survival for HOSU-53 was 60-days compared to vehicle at 26-days in the RPMI-8226 model. We further verified HOSU-53 efficacy using the disseminated MM1.S luciferase CDX model and found a significant prolonged survival in the HOSU-53 cohort (median survival53-days) compared to vehicle (median survival28-days) that was further enhanced with isatuximab combination resulting in superior survival benefit (median survival 69-days). Given the continuous challenge in maintaining a durable response with anti-CD38 therapies due to resistance, we sought to explore additional combination regimens to maximize HOSU-53 efficacy. Currently there is significant clinical interest in CD47 antibody therapy such as magrolimab for both solid tumors and hematological malignancies. Our previous work suggested a strong synergy between CD47 antibodies and HOSU-53 in AML with curative potential (2022 ASH annual meeting abstracts), and hypothesized that we would observe similar synergy with HOSU-53 in combination with anti-CD47 in MM. Indeed, we found complete tumor regression in all mice treated with HOSU-53 + B6.H12 anti-CD47 therapy in the NCI-H929 s.c CDX model and significant prolonged survival and reduced bioluminescence in the NCI-H929 luciferase disseminated CDX model. Together, these two studies validate a potential clinical benefit to explore HOSU-53 + anti-CD47 regimen in MM patients. Furthermore, we observed that calreticulin (pro-phagocytosis signal) was modulated post HOSU-53 in vitro treatment suggesting its role in the observed synergy between HOSU-53 and CD47 blockade therapy. In summary, we show compelling survival benefit for HOSU-53 as a monotherapy which is further enhanced when combined with anti-CD38 or anti-CD47 therapies. HOSU-53 is expected to enter phase 1 clinical trials in 2024 and our data is supportive for its expansion into MM.

Introduction: The use of allogeneic hematopoietic cell transplantation to treat acute myeloid leukemia (AML) has risen in recent years. However, relapse remains the major cause of mortality post-transplant; while graft-versus-host disease (GVHD) - a T cell mediated immunological disorder is the major cause of non-relapse mortality. Dihydroorotate dehydrogenase (DHODH) supports T cell proliferation by playing a critical role in de novo pyrimidine synthesis and oxidative phosphorylation. We hypothesized that alloreactive T cells may rely on increased levels of pyrimidine pools and ATP to support rapid cell proliferation, making DHODH an interesting target to prevent GVHD. Additionally, DHODH inhibition is currently being pursued as a therapeutic option for AML. Therefore, we hypothesize that DHODH inhibition post-transplant will serve a dual purpose - i) target T cell metabolism to reduce GVHD and ii) prevent relapse due to direct anti-leukemic effects, thereby resulting in superior post-transplant outcomes. Methods: We tested the efficacy of a novel DHODH inhibitor (Cmpd 41), a lead clinical candidate, in preventing GVHD and retaining graft-versus-leukemia (GVL) effect. Human T cells isolated from PBMCs were activated with CD3/CD28 Dynabeads ± Cmpd 41. Cell proliferation (flow cytometry) and ATP production (Agilent Seahorse) was assessed. GVHD and GVL was assessedevaluated in a xenogeneic model where irradiated NSG mice received human PBMCS (~17x106 cells) and treated with vehicle or Cmpd 41 (10mg/kg, 2x/week) with addition of MOLM-13 cells (~1x104) for GVL. Splenocytes were harvested for analysis of cytokine production (intracellular flow cytometry). Results: DHODH inhibition with Cmpd 41 significantly reduced T cell proliferation and ATP production from both glycolysis and OXPHOS (fold change: Cmpd 41 vs. control- 0.56 and 0.68 respectively, p<0.01) compared to vehicle. DHODH inhibition significantly improved survival (mean survival: Cmpd 41 vs. vehicle: 56 days vs. 40 days, p<0.01) and reduced clinical scores (Cmpd 41 vs. vehicle: 1.8 vs. 4.2, p<0.05) compared to vehicle in the xenogeneic GVHD model. There was a significant reduction in IFN-γ and TNF-α cytokine producing T cells in the Cmpd 41 treated cohort compared to vehicle (Cmpd 41 vs. vehicle, IFN-γ: 8.33% vs. 31.28%, p<0.01; TNF-α: 1.37% vs. 27.25%, p<0.01). In a GVL model, mice that received both human PBMCs and Cmpd 41 showed decreased tumor growth and improved overall survival over mice given either treatment alone (p<0.01), showing that DHODH inhibition maintains GVL. Conclusion: DHODH inhibition is a novel approach to prevent and mitigate GVHD while retaining GVL effects. Combined with direct anti-leukemic effects, we propose that Cmpd 41 treatment in post-transplant relapse in the setting of past or active graft versus host disease will provide dual treatment of AML and GVHD thereby leading to improved patient outcomes. Citation Format: Kara M. Braunreiter, Lotus Neidemire-Colley, Natalie Sell, Yandi Gao, Sandip Vibhute, Chad Bennett, Ola A. Elgamal, Thomas Goodwin, Erin K. Hertlein, John C. Byrd, Parvathi Ranganathan. DHODH inhibition modulates T cell metabolism reducing GVHD and prevents relapse following allogeneic HCT [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 2335.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

Epithelial ovarian cancer is the most lethal malignancy of the female reproductive tract. A healthy ovary expresses both Estrogen Receptor α (ERα) and β (ERβ). Given that ERα is generally considered to promote cell survival and proliferation, thereby, enhancing tumor growth, while ERβ shows a protective effect against the development and progression of tumors, the activation of ERβ by its agonists could be therapeutically beneficial for ovarian cancer. Here, we demonstrate that the activation of ERβ using a newly developed ERβ agonist, OSU-ERb-12, can impede ovarian cancer cell expansion and tumor growth in an ERα-independent manner. More interestingly, we found that OSU-ERb-12 also reduces the cancer stem cell (CSC) population in ovarian cancer by compromising non-CSC-to-CSC conversion. Mechanistically, we revealed that OSU-ERb-12 decreased the expression of Snail, a master regulator of the epithelial-to-mesenchymal transition (EMT), which is associated with de novo CSC generation. Given that ERα can mediate EMT and facilitate maintenance of the CSC subpopulation and that OSU-ERb-12 can block the transactivity of ERα, we conclude that OSU-ERb-12 reduces the CSC subpopulation by inhibiting EMT in an ERα-dependent manner. Taken together, our data indicate that the ERβ agonist OSU-ERb-12 could be used to hinder tumor progression and limit the CSC subpopulation with the potential to prevent tumor relapse and metastasis in patients with ovarian cancer.

PV007 / #645 Poster Topic: AS02 - Animal Models Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that causes inflammation in many of the body’s tissues, including the skin, joints, lungs, kidneys and heart. This inflammation causes damage to these tissues and produces significant mortality, with most complications in the early stages of the disease involving direct effects on various organs. SLE affects young women between the ages of 15 and 45 years at a 9:1 rate as compared to men. The etiology is complex and remains elusive, however the susceptibility of women during the years in which estrogen levels are at their highest may suggest a significant and critical contribution to the development of SLE. Estrogens are known to have pleiotropic effects on the immune system which is mediated through either estrogen receptor α (ERα), estrogen receptor β (ERβ) or the cell surface G-protein coupled receptor GPER1. ERα has been studied extensively in this context, however ERβ and GPER1 have received much less attention. Previously, our group and others have shown that ERα carrying immune cells are mediators of proinflammatory effects of estrogen more so than cells that lack this receptor. In the pursuit of a drug that is tailored to have a favorable selective estrogenic effect, the OSU Drug Development Institute discovered a novel carborane-based selective estrogen receptor modulator (SERM), WT-IV-012. This ERβ agonist exhibits potent binding of human ERβ (Ki = 2.0 nM) and functional selectivity for ERβ over ERα of at least 200-fold. The work presented herein describes the potential utility of WT-IV-012 in treating SLE in a humanized mouse model of the disease. PBMC isolated from patients with active SLE were adoptively transferred into NSG mice and allowed to expand in vivo for 1 week. The mice were divided into 3 cohorts receiving either vehicle control, prednisone or WT-IV-012 via oral gavage on a daily basis for 5 weeks. Blood samples were taken at baseline, 3 and 5 weeks. Serum was analyzed for circulating cytokines using the MSD human V-PLEX Proinflammatory Panel. At 5 weeks, mice were euthanized, kidneys and hearts were harvested and processed for H&E and IHC histology and urine was collected and tested for proteinuria. Lupus patient PBMCs were isolated and stimulated under various conditions and then flow cytometry was used to identify specific cell types affected by WT-IV-012 and cytokine ELISAs were used to evaluate the cell culture supernatants. WT-IV-012 was as effective as prednisone in suppressing immune cell invasion of the kidney as well as inflammation of the heart. Furthermore, the ERβ agonist demonstrated superior effectiveness in suppressing proinflammatory cytokines as compared to prednisone as well as reducing the proteinuria seen in the vehicle-treated control mice. The in vitro effect of WT-IV-012 confirmed the suppression of IFNγ and TNFα and revealed the cell type specific effects of the drug. WT-IV-012 is an effective inhibitor of the SLE inflammatory process and warrants additional study as a potential therapeutic in patients with SLE.

Background: Glioblastoma (GBM) is the most aggressive primary brain tumor, representing approximately 15% of all primary CNS malignancies. Historically, the occurrence of GBM is higher in male than in female of reproductive age. Usage of exogenous hormones are known to reduce the risk of glioma development and estrogen improves survival in a glioblastoma animal models, suggesting a potential protective role in GBM. Estrogen receptor β (ERβ) may play a role as a tumor suppressor in GBM, and interestingly its expression decreases in higher-grade tumor samples with loss of differentiation. Therefore, a selective ERβ agonist could be a potential therapy against GBM. OSU-ERb-12 is brain penetrant, orally bioavailable ERβ agonist and demonstrates selectivity of ERβ: ERα = 46:1. In this work, we have tested the efficacy of this drug in vitro against glioma cells and in an orthotopic, syngeneic, immune-intact GBM mouse model. Materials & Methods: We tested the efficacy of OSU-ERb-12, a novel selective ERβ agonist against commercial glioma cell lines and patient derived glioma stem like cells, cell proliferation rate via cell titer Glo assay. To evaluate the cell death Annexin-PI assay was performed using flow cytometry. We also tested impact of this drug on cell cycle distribution using flow cytometry-based assay. For in vivo studies, C57BL/6 mice were intracranially implanted with the murine glioma cell line GL261-Luc2 and treated with 30 mg/kg and 100 mg/kg of OSU-ERb-12. Tumor growth was evaluated post implantation using bioluminescent IVIS imaging and animal survival (Kaplan-Meier) was recorded. Results: Treatment with OSU-ERb-12 significantly reduced the cell proliferation rate of commercial cells and patient derived glioma stem cells from various molecular classifications with IC50= 5 µM, 72h. Cells treated with OSU-ERb-12 showed a time and dose dependent enhancement in programmed cell death. Activation of ERβ did not lead to any significant change in cell cycle pattern. Although treatment with 30 mg/kg OSU-ERb-12 did not show any change in survival (median survival 28 days) of tumor bearing mice, a significantly improved median survival was observed for mice dosed at 100 mg/kg (median survival 45 days) in comparison to vehicle (median survival 28 days). Conclusions: Our study demonstrates an important role of ERβ for glioma cell survival as observed by the potent biological effects of OSU-ERb-12 against glioma cell lines and immune intact murine glioma model that supports the potential for targeting the ERβ as a novel therapeutic strategy for treatment of GBM. Citation Format: Pratibha Sharma, Jayeeta Ghose, Christopher Coss, Chad Bennett, Raju R. Raval, Vinay Puduvalli. Preclinical characterization of OSU- ERb-12, a novel ERβ agonist in Glioblastoma [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 1867.

Common FDA approved strategies for hematological malignancies include chemotherapy, targeted therapies, immune therapies, hypomethylating agents, and cell-based therapeutics. However, recurrence often occurs resulting in poor prognosis particularly in acute myeloid leukemia (AML) with first relapse and multiple myeloma (MM) following several relapses based upon more therapies being available. The emergence of dihydroorotate dehydrogenase as an appealing mechanistic target in AML has propelled the development of several clinical candidate DHODH inhibitors (DHODHi) such as brequinar. Here we describe HOSU-53, a novel DHODHi with favorable oral bioavailability (>50% in mouse, rat, and dog) and a pharmacodynamic (PD) biomarker for target engagement, enabling clinical in vivo monitoring of potency and on-target anti-tumor activity. Here we describe the development of a series of DHODHi through a collaboration between Hendrix College and The Ohio State University (OSU). Our initial screen of 18 novel compounds synthesized at Hendrix College identified HOSU-3 as a potent DHODHi with efficacy equivalent to brequinar, however in vivo efficacy in a MOLM-13 AML xenograft model revealed further optimization would be required to compete with leading clinical candidates. Further structure activity relationship studies and lead optimization performed in collaboration with the Drug Development Institute at OSU led to screening over 200 additional analogs and the development of three additional compounds (HOSU-28, -49 and -53). HOSU-53 has subnanomolar biochemical activity against human DHODH in a cell-free enzyme assay, with a cellular IC50 ranging from 2 - 45 nM across a panel of AML cell lines, and 12-42 nM in MM cell lines. While HOSU-53 is highly plasma protein bound, it exhibits a moderate serum shift in assays using human plasma proteins with an IC50 of 457 nM, well below the in vivo Cmax and AUC achieved in rodent and canine pharmacokinetic (PK) studies (Cmax = 79.4-210 μM and AUC = 997-7961 μM-h). Additional absorption, distribution, metabolic and excretion (ADME) and safety studies revealed that HOSU-53 displays very favorable drug-like properties sufficient for development as a pre-clinical candidate. A MOLM-13 xenograft study verified that HOSU-53 was a potential new best in class DHODHi, with efficacy superior to the Bayer clinical candidate. We performed a follow-up study to determine the efficacy of dosing daily at 4, 10, and 20 mg/kg versus 30 mg/kg twice a week. Correlative PK/PD studies were also conducted to measure plasma HOSU-53 and DHO levels at day 1 and 14. We observed a dose linear relationship between HOSU-53 plasma concentration and DHO accumulation. Collectively, our efficacy and PK/PD studies indicate that 10 mg/kg is a highly efficacious and tolerable daily regimen, while higher doses can be safely administered intermittently, and DHO accumulation serves as an important plasma biomarker to identify in vivo therapeutic responses and toxicity. Confirmatory studies in rat subcutaneous MOLM-13 and MV4-11 xenograft models further demonstrated the efficacy of HOSU-53. Our group has previously described synergy between HOSU-53 and decitabine in AML, as well as monoclonal antibodies directed to CD38 in AML and MM. Furthermore, a novel combination between HOSU-53 and CD47 antibody is described in a separate abstract at this current meeting. We expanded our combination studies by testing HOSU-53 with another standard of care, the FLT3 inhibitor gilteritinib, using the MOLM-13 FLT3-ITD mutant xenograft model. NCG mice were enrolled ten days post engraftment to receive daily 4 mg/kg HOSU-53, 30 mg/kg gilteritinib or the combination. Single agent HOSU-53 and gilteritinib prolonged survival (median survival of 37 and 47 days, respectively) while the combination significantly improved survival with 9/10 mice in the combination group remaining on study at day 80. Collectively, we provide a thorough preclinical assessment for HOSU-53 for treatment in hematological malignancy both as a monotherapy and combined with other effective approved therapies including decitabine, monoclonal antibodies and gilteritinib. We have initiated IND enabling studies including GLP toxicity studies planned for late 2022 and have initiated human dose projection models based on our multi-species PK/PD analysis. We anticipate filing an IND application in 2023 for a phase 1 clinical trial in AML and MM.