Primary Patient Cells Research Articles

O-246 The molecular mechanism of miR-96-5p in the pathogenesis and treatment of polycystic ovary syndrome

Abstract Study question Polycystic ovary syndrome, characterized by the androgen excess and arrest of antral follicles, is lacking novel specific diagnostic biomarkers and therapeutic targets. Summary answer Study of the molecular mechanisms of microRNAs in the etiology and its potential applications in PCOS provides the insight. What is known already Some of molecular mechanisms of microRNAs have been demonstrated involved in the etiology of PCOS Study design, size, duration Clinically, we collected the samples of serum, follicular fluid (FF) and primary human granulosa cells (hGCs) of PCOS patients (n = 70) vs. non-PCOS women (n = 60). Experimentally, we carried out studies with 3-types of induced PCOS-like mice. Participants/materials, setting, methods By analysing the expression levels of miR-96-5p in serum, follicular fluid (FF) and primary human granulosa cells (hGCs) of PCOS patients (n = 70) vs. non-PCOS women (n = 60). as well as in ovaries from 3-types of induced PCOS-like mice. Main results and the role of chance Clinically, we demonstrated that the elevated circulating miR-96-5p levels were significantly correlated with the PCOS disordered endocrine clinical features, and the area under the curve of receiver operating characteristic was 0.8344, with 75.71% specificity and 80% sensitivity. Mechanically, we identified miR-96-5p as an androgen-regulated miRNA that directly targets the forkhead transcription factor FOXO1. Inhibition of miR-96-5p decreased estrogen synthesis, while decreasing the cell proliferation index of KGN via regulating the expression of FOXO1 and its downstream genes. Inversely, inhibition of FOXO1 abrogated the effect of miR-96-5p on estrogen synthesis and proliferation index. Of note, ovarian intra-bursal injection of miR-96-5p agomir rescued the phenotypes of dehydroepiandrosterone-induced PCOS like mice. Limitations, reasons for caution This study needs a large-scale clinical investigation to confirm the specificity and sensitivity of miR-96-5p as a clinical biomaker. Wider implications of the findings In conclusion, our results clarified a vital role of miR-96-5p in the pathogenesis of PCOS and might serve as a novel diagnostic biomarker and therapeutic target for PCOS. Trial registration number no

BV6, AN ANTAGONIST OF INHIBITOR OF APOPTOSIS PROTEINS, SENSITIZES RESISTANT MANTLE CELL LYMPHOMA CELLS TO VENETOCLAX

Introduction: Although Bruton’s tyrosine kinase inhibitors (BTKi) and CAR T-cell therapy have achieved remarkable efficacy in the treatment of mantle cell lymphoma (MCL), it remains incurable due to the inevitable development of therapeutic resistance. The BCL-2 inhibitor venetoclax is effective in many relapsed MCL patients, including those who progressed on BTKi, but subsequent relapse to venetoclax yields poor clinical outcomes. Hence, novel therapeutics and combination therapies are required to tackle the clinical challenges. Here, we investigated BV6, an antagonist of inhibitor of apoptosis proteins (IAPs), alone and in combination with venetoclax in multiple therapy-resistant MCL models. Methods: We performed single-cell RNA-seq to assess gene expression in ibrutinib-sensitive, ibrutinib-resistant, and dual ibrutinib-/CAR T-resistant MCL patients. Cell lines with acquired resistance to ibrutinib or venetoclax, as well as primary tumor cells from patients who relapsed after multiple therapies, were used as models of MCL resistance. CellTiter-Glo® luminescent assay was used to measure cell viability; annexin V/PI staining was used to measure the induction of apoptosis; and western blotting was used to assess IAP and apoptosis signaling protein expression across MCL cell lines after drug treatment. Results: The single-cell RNA-seq analysis demonstrated that IAP gene expression was not influenced by patient response to ibrutinib and CAR T-cell therapy, and western blotting showed that IAP proteins were broadly expressed across all MCL cell lines. To determine the on-target effects of BV6, we treated JeKo-1, JeKo-IBN-R, Mino, and Mino-VEN-R cells with BV6 and examined the expression of IAPs. BV6 treatment for 4h markedly reduced c-IAP1 and c-IAP2 expression, and moderately reduced XIAP expression. BV6 inhibited cell viability in both ibrutinib- and venetoclax-resistant cell lines, and BV6 treatment for 24 h dose-dependently induced apoptosis in MCL cells, including not only cell lines with acquired resistance but also patient primary cells, independent of their resistance to ibrutinib, venetoclax, and CAR T-cell therapy. We observed a striking and significant synergy in the cell viability assay when ibrutinib- or venetoclax-resistant cells were treated with BV6 combined with venetoclax (combination index < 1), and a dramatic enhancement of apoptosis (p < 0.001). The combination eradicated c-IAP1 and c-IAP2 expression, while XIAP was synergistically diminished. This was accompanied by strong caspase-3 activation and PARP cleavage. This combination therapy is currently under evaluation in venetoclax-resistant mouse model in vivo. Conclusions: Our findings indicate that a combination of IAP antagonists with apoptosis inducers can be exploited as a rational strategy to overcome therapeutic resistance in MCL. This study was supported by philanthropic funds from the Kimmel Research fund and various donor funds. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Delivery of Engineered Primary Tumor-Derived Exosomes Effectively Suppressed the Colorectal Cancer Chemoresistance and Liver Metastasis.

Liver metastasis is one of the major causes of colorectal cancer (CRC)-related morbidity and mortality. Delivering small interfering RNAs (siRNAs) or noncoding RNAs has been reported as a promising method to target liver metastasis and chemoresistance in CRC. Here, we report a noncoding RNA delivery system using exosomes derived from primary patient cells. Coiled-coil domain-containing protein 80 (CCDC80) was strongly associated with CRC liver metastasis and chemoresistance, a finding validated by bioinformatic analysis and clinical specimens. Silencing CCDC80 significantly increased sensitivity to chemotherapy agents in OXA-resistant cell lines and a mouse model. The primary cell-derived exosome delivery system was designed to simultaneously deliver siRNAs targeting CCDC80 and increase chemotherapy sensitivity in the distant CRC liver metastasis mouse models and patient-derived xenograft mouse models. We further validated the antitumor effect in an ex vivo model of chemoresistant CRC organoids and a patient-derived organoid xenograft model. Tumor-bearing mice treated with the siRNA-delivering exosomes and hepatectomy showed ideal overall survival. Our results provide a therapeutic target and represent a possible therapeutic alternative for patients with CRC and distant metastasis and in cases of chemoresistance.

A New Registry for Genetic Diagnosis of Inborn Errors of Immunity within the Military Health System

Genotypic definition of monogenic inborn errors of immunity (IEIs) continues to accelerate with broader access to next generation sequencing, underscoring this aggregated group of disorders as a major health burden impacting both civilian and military populations. At an estimated prevalence of 1 in 1200 individuals, IEIs affect ~8,000 patients within the Military Health System (MHS). Despite access to targeted gene/exome panels at military treatment facilities, most affected patients never receive a definitive genetic diagnosis that would significantly improve clinical care. To address this gap, we established the first registry of IEI patients within the MHS with the goal of identifying known and novel pathogenic genetic defects to increase diagnosis rates and enhance clinical care. Using the registry, a research protocol was opened in July 2022. Since July we have enrolled 75 IEI patients encompassing a breadth of phenotypes including severe and recurrent infections, bone marrow failure, autoimmunity/autoinflammation, atopic disease, and malignancy. Enrolled patients provide blood and bone marrow samples for whole genome, ultra-deep targeted panel and comprehensive transcriptome sequencing, plus cryopreservation of peripheral blood mononuclear cells for future functional studies. We are also implementing and developing analytical methods for identifying and interrogating non-coding and structural variants. Suspected pathogenic variants are adjudicated by a clinical molecular geneticist using state-of-the-art analysis pipelines. These analyses subsequently inform in vitro experiments to validate causative mutations using cell reporter systems and primary patient cells. Clinical variant validation and return of genetic results are planned with genetic counseling provided. As a proof of principle, this integrated genetic evaluation pipeline revealed a novel, candidate TLR7 nonsense variant in two adolescent brothers who both endured critical COVID-19 pneumonia, requiring mechanical ventilation and extracorporeal membrane oxygenation. Our protocol is therefore poised to greatly enrich clinical genetics resources available in the MHS for IEI patients, contributing to better diagnosis rates, informed family counseling, and targeted treatments that collectively improve the health and readiness of the military community. Moreover, our efforts should yield new mechanistic insights on immune pathogenesis for a broad variety of known and novel IEIs.

Abstract 288: Development Of A Phenotypic Screen With 56 Marfan Syndrome Patient-derived Fibroblasts

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the extracellular matrix gene coding for fibrillin-1 (FBN1). MFS leads to aortic aneurysms as key feature. Due to the high genetic diversity within the MFS patient population, genotype-phenotype predictions on disease progression and response to therapy remain challenging. Furthermore, current preclinical murine models represent only a subset of FBN1 mutations, which limits translational studies. In this study, we developed a preclinical model using primary cells of MFS patients that represent genetic and clinical variability in order to develop improved drug strategies. Human primary fibroblasts were isolated from skin biopsies derived from MFS patients (n=56) participating in the COMPARE trial. Human aortic smooth muscle cells (AoSMCs) were isolated from aortic tissue derived from MFS patients undergoing prophylactic aortic surgery (n=4). Long-term cultures in 384-well plates were assessed for the extracellular matrix and disease associated pathways. Analysis of all MFS fibroblasts showed either a reduced or abolished fibrillin-1 extracellular matrix network. Moreover, substantially reduced secretion of fibrillin-1 and asprosin in the culture medium was measured, as compared to healthy control fibroblasts. MFS fibroblasts with an abolished fibrillin-1 matrix were associated with patients with more rapid aortic root diameter growth. Exploring disease-relevant pathways revealed reduced nuclear localization of transcription factor KLF4 in MFS fibroblasts, which associated with the level of fibrillin-1 matrix. As a marker for myofibroblasts, α-smooth muscle actin (ASMA) staining was increased in these MFS fibroblasts. These findings were validated in AoSMC cultures, showing similar MFS fibrillin-1 matrix, nuclear KLF4, and ASMA expression profiles. In conclusion, phenotypic screening of patient-derived cells contributes to improved understanding of disease development and is valuable as preclinical tool for risk assessment and drug development for MFS and related conditions.

Microphysiological head and neck cancer model identifies novel role of lymphatically secreted monocyte migration inhibitory factor in cancer cell migration and metabolism

Regional metastasis of head and neck cancer (HNC) is prevalent (approximately 50% of patients at diagnosis), yet the underlying drivers and mechanisms of lymphatic spread remain unclear. The complex tumor microenvironment (TME) of HNC plays a crucial role in disease maintenance and progression; however, the contribution of the lymphatics remains underexplored. We created a primary patient cell derived microphysiological system that incorporates cancer-associated-fibroblasts from patients with HNC alongside a HNC tumor spheroid and a lymphatic microvessel to create an in vitro TME platform to investigate metastasis. Screening of soluble factor signaling identified novel secretion of macrophage migration inhibitory factor (MIF) by lymphatic endothelial cells conditioned in the TME. Importantly, we also observed patient-to-patient heterogeneity in cancer cell migration similar to the heterogeneity observed in clinical disease. Optical metabolic imaging at the single cell level identified a distinct metabolic profile of migratory versus non-migratory HNC cells in a microenvironment dependent manner. Additionally, we report a unique role of MIF in increasing HNC reliance on glycolysis over oxidative phosphorylation. This multicellular, microfluidic platform expands the tools available to explore HNC biology in vitro through multiple orthogonal outputs and establishes a system with enough resolution to visualize and quantify patient-to-patient heterogeneity.

Combination Treatment Targeting mTOR and MAPK Pathways Has Synergistic Activity in Multiple Myeloma

Multiple myeloma (MM) is an incurable, malignant B cell disorder characterized by frequent relapses and a poor prognosis. Thus, new therapeutic approaches are warranted. The phosphatidylinositol-3-kinase (PI3K) pathway plays a key role in many critical cellular processes, including cell proliferation and survival. Activated PI3K/AKT (protein kinases B)/mTOR (mammalian target of rapamycin) signaling has been identified in MM primary patient samples and cell lines. In this study, the efficacy of PI3K and mTOR inhibitors in various MM cell lines representing three different prognostic subtypes was tested. Whereas MM cell lines were rather resistant to PI3K inhibition, treatment with the mTOR inhibitor temsirolimus decreases the phosphorylation of key molecules in the PI3K pathway in MM cell lines, leading to G0/G1 cell cycle arrest and thus reduced proliferation. Strikingly, the efficacy of temsirolimus was amplified by combining the treatment with the Mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Our findings provide a scientific rationale for the simultaneous inhibition of mTOR and MEK as a novel strategy for the treatment of MM.

849 Twin prime editing for restoring type VII collagen expression in primary recessive dystrophic epidermolysis patient cells

849 Twin prime editing for restoring type VII collagen expression in primary recessive dystrophic epidermolysis patient cells

Abstract 5866: Mavorixafor enhances apoptosis of tumor cells treated with ibrutinib or venetoclax in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia

Abstract Despite advances in therapies for B-cell lymphoma, patients may still develop resistance and often relapse. Contributing factors may include interactions between lymphoma cells (LCs) or leukemia cells and bone marrow microenvironment (BMM). Overexpression of CXCR4WT and its ligand CXCL12 promote LC-BMM interactions, contributing to disease severity in lymphomas or leukemia such as diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). Mavorixafor is an investigational oral CXCR4 antagonist being evaluated in combination with the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in patients with Waldenström’s macroglobulinemia (WM). In preclinical models of MYD88L265P CXCR4WT WM, mavorixafor restored sensitivity of WM cells to B-cell-targeted therapies in coculture of WM cells and bone marrow stromal cells (BMSCs); however, the effects of mavorixafor on other lymphomas with CXCR4WT have not been evaluated. We report the effects of mavorixafor alone or in combination with B-cell─targeted therapies on LCs in an in vitro preclinical model and assess the role of BMM in the pathogenesis of B-cell lymphomas in an in vitro coculture model of LCs and BMSCs. Bioinformatic analysis of publicly available gene expression datasets GSE11318 and GSE32918 showed patients with DLBCL and CXCR4hi have significantly inferior survival probability compared with those with CXCR4low (P&amp;lt;.001 and P&amp;lt;.05, respectively). In vitro studies assessing effects of targeting CXCR4 with mavorixafor showed apoptosis of CXCR4+ LCs OCILY19 (DLBCL), MEC1 (CLL), MINO (MCL), and DOHH2 (FL) cells increased by ≈10%─60%. The apoptosis of LCs was further enhanced by ≈5%─60% when treated with mavorixafor in combination with ibrutinib or venetoclax compared to ibrutinib or venetoclax alone. Coculture of LCs with BMSCs reduced the sensitivity of LCs to apoptosis-inducing effects of ibrutinib and venetoclax. Combining mavorixafor with ibrutinib or venetoclax restored sensitivity of LCs to apoptosis-inducing effects of the tested B-cell─targeted inhibitors. Mavorixafor also inhibited migration of LCs toward CXCL12, suggesting prevention of the homing of LCs to protective niche. Overall, our findings suggest the contribution of CXCR4WT to pathogenicity of LCs. This is the first in vitro study to show that reduced sensitivity of LCs against B-cell-targeted therapies conferred by BMSCs can be overcome by inhibition of the CXCL12-CXCR4 axis with mavorixafor. Our study provides supporting evidence for further exploration of mavorixafor alone or in combination with other B-cell-targeted therapies in the treatment of lymphomas and leukemias. Further studies using additional LC lines and/or primary patient cells are warranted to support these findings. Citation Format: Tom Kruitwagen, Thalia Martins Rebelo, Barbara Maierhofer, Gerwin Heller, Halenya Monticelli, Arthur G. Taveras, Chi Nguyen. Mavorixafor enhances apoptosis of tumor cells treated with ibrutinib or venetoclax in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5866.

Abstract 2677: ROCK inhibitors synergize with the BCL2 inhibitor venetoclax for treatment of AML

Abstract Acute Myeloid Leukemia (AML) is a rare but catastrophic hematological cancer with a poor prognosis, high mortality, and relapse rates. Several new agents targeting specific genes or cell survival pathways have been recently approved by the FDA. AML treatment with single agents often results in resistance; therefore, combination therapies may provide more durable and effective responses. Rho kinases (ROCK1/2) are aberrantly expressed in AML patient cells, and ROCK1 has emerged lately as a potential therapeutic target for AML treatment. Previously, our group developed a new ROCK inhibitor DJ4 as an effective inducer of AML cell death. Our initial screening of DJ4 with AML standard therapy (cytarabine, daunorubicin, azacytidine, venetoclax) showed promising synergy between DJ4 and the BCL2 (B-cell lymphoma-2) inhibitor venetoclax (VEN). Enrichment of ROCK pathway in recent CRISPR drop-out screening after VEN treatment in MOLM-13 cells further validates the synergistic cooperation between ROCK and BCL2 pathways. Though VEN is widely used in hematological malignancies, its clinical efficacy is limited due to the development of resistance. Thus, we sought to investigate the novel strategy of combining VEN with ROCK inhibitors (ROCKi) for AML treatment. The combination of VEN with ROCKi (Fasudil, Y-27632, GSK269962A, DJ4) exhibited synergetic cytotoxicity in naïve (MOLM-13, MV4-11) and VEN-resistant (MV4-11/VENR, HL-60, OCI-AML3, U937) human AML cell lines. Flow cytometric analysis of Annexin V and cellular ROS levels in MV4-11 (naïve and VENR) cells upon co-treatment with sub-lethal doses of VEN and ROCKi demonstrated a significant (p&amp;lt;0.05) increase in the apoptosis and oxidative stress compared to single agents. Treatment of MV4-11 cells with VEN+ROCKi enhanced the activity of several caspases in the MultiCaspase assay. Furthermore, the colony-forming ability of AML cell lines and primary AML patient cells was significantly reduced with the combination treatment, while single agents exhibited minimal effects. MV4-11 (naïve and VENR) cells displayed alterations in the levels of critical apoptosis regulators (Bcl2, Bax, Mcl1, cleaved caspase-3) upon co-treatment in immunoblotting analysis. Additionally, MV4-11-Luc xenograft mice exhibited a significant reduction in the average leukemic burden after combination therapy relative to single agents (p&amp;lt;0.05). Altogether, our in vitro and in vivo findings established a synergetic interaction between VEN and ROCKi to effectively inhibit AML cell progression and overcome the VEN resistance (inherent or acquired). These results indicate that therapy co-targeting BCL2 and ROCK pathways exerted antileukemic activity by inducing apoptosis and may be particularly effective for AML treatment. Further studies are ongoing to explore the precise molecular mechanism of action of the VEN+ROCKi combination and its efficacy in pre-clinical animal models. Citation Format: Upendarrao Golla, Satyam Patel, Diwakar B. Tukaramrao, Jeremy Hengst, Juliana Restrepo, Shantu Amin, Dhimant Desai, Sinisa Dovat, David Claxton, Arati Sharma. ROCK inhibitors synergize with the BCL2 inhibitor venetoclax for treatment of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2677.

Abstract 5798: Ex vivo culturing of primary AML cells facilitates the study of developmental therapeutics

Abstract Studies with acute myeloid leukemia (AML) cell lines have limited potential as they tend to acquire mutations, thus, do not represent typical leukemias found in patients. Ex vivo culturing of primary human AML patient cells is therefore of interest; however, the long-term culture of these cells has been limited by rapid differentiation and loss of clonogenicity. Previously, different growth factors and stromal cells were implicated in the short-term culturing of primary cells. Thus, we aimed to evaluate culture conditions using a cytokine cocktail, allowing long-term expansion and phenotypic maintenance of primary AML cells in vitro. Different culture conditions were constituted by combination of cytokines (SCF, FLT-3L, TPO, IL-6, IL-3, GCSF, GM-CSF) and small molecules (SR1, UM729) in serum-free StemSpan SFEM media. The ability of different growth mediums to support the expansion and clonogenicity of 6 primary AML cells (collected from blood/bone marrow) over two weeks was assessed using cell counts and colony formation assays. Additionally, cell surface markers (hCD34, hCD45, hCD123, hCD117) were assessed by flow cytometry during the culture period. All tested cultured conditions exhibited successful cell proliferation (4-6-fold expansion) and survival of primary AML cells over two weeks of culture. In addition, the growth media successfully supported 3.5-fold CD34+ stem cell expansion. Growth mediums with early-acting cytokines (SCF, FLT-3L, TPO, and IL-6) and small molecules resulted in phenotypic maintenance of cell surface markers (CD34, CD123) and secondary clonogenicity of primary AML cells over the culture period. The addition of late-acting cytokines (IL-3, GCSF, and GM-CSF) showed a higher growth rate but negatively affected the cell surface markers and clonogenicity. Secondary colony counts (day-9 culture) showed a significant positive correlation with CD34 expression levels. Thus, CD34 levels could serve as a potential surrogate marker for the clonogenicity of primary AML cells. Furthermore, we tested the engraftment ability of two primary AML cells grown in 3 of our promising culture conditions (Day 4, Day 8) in xenograft mice. Flow cytometry analysis on peripheral blood and tissues (spleen and bone marrow) from xenograft mice showed successful engraftment of primary cells cultured for 8 days. On an application note, the optimal condition from our results was used to study the effects of ceramide nanoliposomes (CNL) in AML patient cells. Interestingly, the AML patient cells treated for two weeks with IC15 and IC30 doses of CNL led to cell cycle arrest, reduced CD34 expression, and clonogenicity relative to control cells. The CNL findings from primary cells are different from that of cell lines cultured under the same conditions. Thus, our optimal growth conditions may facilitate researchers to understand AML biology and study various therapeutics under development using primary AML cells. Citation Format: Satyam Patel, Upendarrao Golla, Arati Sharma, David Claxton. Ex vivo culturing of primary AML cells facilitates the study of developmental therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5798.

Abstract 1679: Unraveling the function of TRAF2 and NCK interacting kinase (TNIK) in high-grade serous ovarian cancer

Abstract The large majority of high grade serous ovarian cancer (HGSOC) patients will eventually become resistant to platinum-based chemotherapy, highlighting the urgent need to develop novel therapeutics targeting recurrent, platinum-resistant disease. To study this, our lab generated two patient derived platinum-resistant tumor spheres from platinum-sensitive primary patient cells to better understand the biology behind recurrent disease and identify novel therapeutic targets. Utilizing an artificial intelligence (AI) powered model, we screened a library that consisted of compounds predicted to target known or postulated resistance mechanisms and involved a novel therapeutic target. From this screen, we identified a TNIK kinase inhibitor as the only compound to effectively kill HGSOC spheres while sparing non-malignant fallopian tube cells. TNIK plays a key role in Wnt pathway activation, which our lab has previously shown to be critical in driving cancer stemness and epithelial ovarian cancer platinum resistance. Additionally, patient data shows that TNIK is amplified in ovarian cancer more than any other cancer type and is highly activated in ovarian cancer patients. We found that this compound was able to decrease overall Wnt activity in our platinum-resistant cells and could resensitize our platinum-resistant cells to cisplatin. This work demonstrates the abilities of using AI to expedite the drug discovery process as well as uncover novel therapeutic targets which may lead to future therapeutic strategies for HGSOC patients who no longer respond to standard of care platinum chemotherapy. Citation Format: Noah Puleo, Michele Cusato, Dylan Carvette, Chantelle Husdon, Analisa DiFeo. Unraveling the function of TRAF2 and NCK interacting kinase (TNIK) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1679.

Mechanism of lnRNA-ICL involved in lung cancer development in COPD patients through modulating microRNA-19-3p/NKRF/NF-κB axis

The incidence of lung cancer (LC) in chronic obstructive pulmonary disease (COPD) patients is dozens of times higher than that in patients without COPD. Elevated activity of nuclear factor-k-gene binding (NF-κB) was found in lung tissue of patients with COPD, and the continuous activation of NF-κB is observed in both malignant transformation and tumor progression of LC, suggesting that NF-κB and its regulators may play a key role in the progression of LC in COPD patients. Here, we report for the first time that a key long non-coding RNA (lncRNA)-ICL involved in the regulation of NF-κB activity in LC tissues of COPD patients. The analyses showed that the expression of ICL significantly decreased in LC tissues of LC patients with COPD than that in LC tissues of LC patients without COPD. Functional experiments in vitro showed that exogenous ICL only significantly inhibited the proliferation, invasion and migration in primary tumor cells of LC patients with COPD compared to LC patients without COPD. Mechanism studies have shown that ICL could suppress the activation of NF-κB by blocking the hsa-miR19-3p/NKRF/NF-κB pathway as a microRNA sponge. Furthermore, In vivo experiments showed that exogenous ICL effectively inhibited the growth of patient-derived subcutaneous tumor xenografts (PDX) of LC patients with COPD and significantly prolonged the survival time of tumor-bearing mice. In a word, our study shows that the decrease of ICL is associated with an increased risk of LC in patients with COPD, ICL is not only expected to be a new therapeutic target for LC in COPD patients, but also has great potential to be used as a new marker for evaluating the occurrence, severity stratification and prognosis of LC in patients with COPD.

Therapy-Resistant Acute Myeloid Leukemia Stem Cells Are Resensitized to Venetoclax + Azacitidine by Targeting Fatty Acid Desaturases 1 and 2.

Recent advances in targeting leukemic stem cells (LSCs) using venetoclax with azacitidine (ven + aza) has significantly improved outcomes for de novo acute myeloid leukemia (AML) patients. However, patients who relapse after traditional chemotherapy are often venetoclax-resistant and exhibit poor clinical outcomes. We previously described that fatty acid metabolism drives oxidative phosphorylation (OXPHOS) and acts as a mechanism of LSC survival in relapsed/refractory AML. Here, we report that chemotherapy-relapsed primary AML displays aberrant fatty acid and lipid metabolism, as well as increased fatty acid desaturation through the activity of fatty acid desaturases 1 and 2, and that fatty acid desaturases function as a mechanism of recycling NAD+ to drive relapsed LSC survival. When combined with ven + aza, the genetic and pharmacologic inhibition of fatty acid desaturation results in decreased primary AML viability in relapsed AML. This study includes the largest lipidomic profile of LSC-enriched primary AML patient cells to date and indicates that inhibition of fatty acid desaturation is a promising therapeutic target for relapsed AML.

PRAME and CTCFL-reactive TCRs for the treatment of ovarian cancer.

Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2'-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.

HSV1716 Prevents Myeloma Cell Regrowth When Combined with Bortezomib In Vitro and Significantly Reduces Systemic Tumor Growth in Mouse Models

Multiple myeloma remains largely incurable due to refractory disease; therefore, novel treatment strategies that are safe and well-tolerated are required. Here, we studied the modified herpes simplex virus HSV1716 (SEPREHVIR®), which only replicates in transformed cells. Myeloma cell lines and primary patient cells were infected with HSV1716 and assessed for cell death using propidium iodide (PI) and Annexin-V staining and markers of apoptosis and autophagy by qPCR. Myeloma cell death was associated with dual PI and Annexin-V positivity and increased expression of apoptotic genes, including CASP1, CASP8, CASP9, BAX, BID, and FASL. The combination of HSV1716 and bortezomib treatments prevented myeloma cell regrowth for up to 25 days compared to only transient cell growth suppression with bortezomib treatment. The viral efficacy was tested in a xenograft (JJN-3 cells in NSG mice) and syngeneic (murine 5TGM1 cells in C57BL/KaLwRijHsd mice) systemic models of myeloma. After 6 or 7 days, the post-tumor implantation mice were treated intravenously with the vehicle or HSV1716 (1 × 107 plaque forming units/1 or 2 times per week). Both murine models treated with HSV1716 had significantly lower tumor burden rates compared to the controls. In conclusion, HSV1716 has potent anti-myeloma effects and may represent a novel therapy for multiple myeloma.

Upregulated estrogen receptors impairs myogenesis and elevates adipogenesis related factor levels in the paravertebral muscles of patients with idiopathic scoliosis

The prevalence of idiopathic scoliosis (IS) is 2–3% worldwide and is more common in girls. Estrogen receptors (ERs) is supposed to be related to sex differences and development of IS. Meanwhile, paravertebral muscle (PVM) abnormalities play important roles in the pathogenesis of IS. But the changes of ERs between the PVMs from IS patients and controls, and the mechanism by which ERs may affect IS patients remain unclear. Thus, the expression levels of ERs, myogenesis regulator (MYOG) and adipogenesis related factors (CEBPA, PPARγ, FABP4), as well as morphological changes in the PVMs and primary skeletal muscle mesenchymal progenitor cells (hSM-MPCs) of IS patients and controls were investigated. Increased expression levels of ERs and CEBPA, PPARγ, FABP4, together with severe myofiber necrosis and fat infiltration, were found in the PVMs of IS patients. Meanwhile, upregulated ERs, FABP4 and CEBPA, downregulated MYOG and impaired myogenesis were also revealed in the hSM-MPCs of IS patients compared with those of controls. Upregulation of ERs inhibited myogenesis but increased expression of CEBPA and FABP4 in C2C12 myoblasts. Nevertheless, treatment of ER antagonist increased expression of MYOG, enhanced myogenesis and decreased expression of CEBPA and FABP4 in skeletal muscle cells of IS patients. Therefore, our study suggested that PVMs specific upregulation of ERs could impair myogenesis and increase the expression of adipogenesis related factors, further leading to PVMs abnormalities in IS patients.

The "3Ds" of Growing Kidney Organoids: Advances in Nephron Development, Disease Modeling, and Drug Screening.

A kidney organoid is a three-dimensional (3D) cellular aggregate grown from stem cells in vitro that undergoes self-organization, recapitulating aspects of normal renal development to produce nephron structures that resemble the native kidney organ. These miniature kidney-like structures can also be derived from primary patient cells and thus provide simplified context to observe how mutations in kidney-disease-associated genes affect organogenesis and physiological function. In the past several years, advances in kidney organoid technologies have achieved the formation of renal organoids with enhanced numbers of specialized cell types, less heterogeneity, and more architectural complexity. Microfluidic bioreactor culture devices, single-cell transcriptomics, and bioinformatic analyses have accelerated the development of more sophisticated renal organoids and tailored them to become increasingly amenable to high-throughput experimentation. However, many significant challenges remain in realizing the use of kidney organoids for renal replacement therapies. This review presents an overview of the renal organoid field and selected highlights of recent cutting-edge kidney organoid research with a focus on embryonic development, modeling renal disease, and personalized drug screening.

P041 The upregulation of tissue factor in colonic mesenchymal cells in active ulcerative colitis is associated with severe and resistant to treatment disease, but tofacitinib has not effect on it

Abstract Background Tissue factor (TF) plays an important role on blood clotting and the risk of thromboembolic events is increased in ulcerative colitis (UC). Tofacitinib, recently introduced in UC therapeutics, may further increase the risk. To delineate thrombosis pathophysiology in this context, TF expression in primary human colonic mesenchymal cells (PHCMC) of patients with active UC was correlated with clinical parameters, serum markers of inflammation and endoscopy. We then treated those PHCMC with tofacitinib with or without cytokines to better mimic the in vivo milieu they are exposed to. Methods PHCMC from endoscopic biopsies of the inflamed mucosa of 10 UC patients with an endoscopic Mayo score ≥2 were treated with all major T helper (Th)1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or T regulatory (Treg; TGF-β, IL-10) cytokines with or without tofacitinib. Cells were lysed, RNA was isolated and reverse-transcribed to cDNA. TF and RPL4 (housekeeping) cDNAs were quantified with real-time PCR. Wilcoxon and Mann-Whitney U tests were used to compare TF ΔΔCT for paired or unpaired values, respectively, and Spearman’s rho to correlate TF ΔΔCT with scale clinical variables and continuous laboratory values. Results Increased TF mRNA abundance in PHCMC was correlated to increased partial Mayo score (Spearman’s rho 0.661, p &amp;lt; 0.044), reduced serum albumin (Spearman’s rho -0.723, p &amp;lt; 0.05) and to more severe endoscopic lesions: cells originating from UC patients of an endoscopic Mayo score 3 expressed 20 times more TF than those from an endoscopic Mayo score 2 (p &amp;lt; 0.017; Figure 1). Moreover, PHCMC from difficult-to-treat patients, defined as requiring &amp;gt;1 biologics, expressed 9 times more TF (Figure 2). On the other hand, treatment with tofacitinib did not upregulate TF (Figure 3). PHCMC expressed receptors and responded to treatment with all major Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or Treg (TGF-β, IL-10) cytokines by further upregulating TF by 5.5-7.5 times (p 0.001-0.035). IL-13 had the maximal effect (Figure 4). Even when tofacitinib was added together with the aforementioned cytokines, it did not further upregulate TF; instead, it tended to partially inhibit their effects. For example, it decreased upregulation of TF by IL-13 by 40%. Conclusion The expression of TF mRNA in active UC is significantly associated with clinically and endoscopically severe disease and resistance to treatment. Tofacitinib per se does not increase TF. Instead, it may limit the upregulating effect of pro-inflammatory cytokines.