OBJECTIVES/GOALS: Leigh Syndrome, French Canadian-Type (LSFC) is a neurometabolic disorder caused by mutation of mitochondria-related gene, LRPPRC. White matter lesions and demyelination in central nervous system are common in LSFC. LRPPRC is enriched in myelinating glial cells, yet its role is not known. Our goal is to elucidate its mechanistic role in myelination. METHODS/STUDY POPULATION: We crossed C57BL/6N mice bearing a LRPPRC-loxP allele with mice bearing a Plp-CreERT2 allele. Mice with the Plp-CreERT2 allele expresses a tamoxifen-inducible Cre under the control of the Plp promoter, which drives expression in oligodendrocytes. Using these strains, we can target the deletion of LRPPRC, via tamoxifen injection, in both newly formed myelin and mature myelin. Plp-CreERT2; LRPPRCL/L (LRPPRC-KO) or control littermate mice will be injected for LRPPRC deletion at developmental and maturation stages of myelin. Immunofluorescence and electron microscopy of isolated brain tissues will be used for myelin integrity analysis. Cognitive functions of the mice will be measured via behavioral tests. Lastly, we will submit tissues for lipidomic analyses to observe any lipid metabolite variation. RESULTS/ANTICIPATED RESULTS: Behavioral and motor defects would be expected in LRPPRC-KO mice performing in cognitive function tasks across myelin maturation stages. Electron microscopy-based structure analysis of optic nerve, corpus callosum, and spinal cord should reveal thin or loss of myelin on the axons of LRPPRC-KO compared to control. Immunofluorescence staining of major myelin structural proteins, including myelin proteolipid protein (PLP), myelin basic protein (MBP), and myelin-associated glycoprotein (MAG) would be expected have lower levels in LRPPRC deficient tissues. Since myelin is a lipid-rich species, we would also expect lipid concentrations to be affected. LRPPRC-KO lipidomic analyses of myelin-related lipids should depict lower levels in comparison to control, which would imply dysfunctional lipid metabolism. DISCUSSION/SIGNIFICANCE: There are limited studies in ameliorating neural deficits caused by LS and LSFC. Successful completion of this project would help elucidate the functions of LRPPRC in myelination and lipid metabolism and potentially provide insights for developing novel therapeutic strategies for alleviating the demyelination and neural deficits in LSFC.

Background Efficient DNA repair in response to standard chemo and radiation therapies often contributes to glioblastoma (GBM) therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double-strand break (DSB) repair and a combination of KDM1A inhibitor and temozolomide (TMZ) in vitro and in vivo using patient-derived glioma stem cells (GSCs). Methods Brain bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. The effect of a combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, western blot, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo. Results TCGA analysis showed KDM1A is highly expressed in TMZ-treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and self-renewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the blood-brain barrier. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ-mediated DNA damage. A combination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice. Conclusions Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

Ovarian cancer (OCa) is the most lethal gynecologic cancer. Emerging data indicates that estrogen receptor beta (ERβ) functions as a tumor suppressor in OCa. Lysine-specific histone demethylase 1A (KDM1A) is an epigenetic modifier that acts as a coregulator for steroid hormone receptors. However, it remain unknown if KDM1A interacts with ERβ and regulates its expression/functions in OCa. Analysis of TCGA data sets indicated KDM1A and ERβ expression showed an inverse relationship in OCa. Knockout (KO), knockdown (KD), or inhibition of KDM1A increased ERβ isoform 1 expression in established and patient-derived OCa cells. Further, KDM1A interacts with and functions as a corepressor of ERβ, and its inhibition enhances ERβ target gene expression via alterations of histone methylation marks at their promoters. Importantly, KDM1A-KO or -KD enhanced the efficacy of ERβ agonist LY500307, and the combination of KDM1A inhibitor (KDM1Ai) NCD38 with ERβ agonist synergistically reduced the cell viability, colony formation, and invasion of OCa cells. RNA-seq and DIA mass spectrometry analyses showed that KDM1A-KO resulted in enhanced ERβ signaling and that genes altered by KDM1A-KO and ERβ agonist were related to apoptosis, cell cycle, and EMT. Moreover, combination treatment significantly reduced the tumor growth in OCa orthotopic, syngeneic, and patient-derived xenograft models and proliferation in patient-derived explant models. Our results demonstrate that KDM1A regulates ERβ expression/functions, and its inhibition improves ERβ mediated tumor suppression. Overall, our findings suggest that KDM1Ai and ERβ agonist combination therapy is a promising strategy for OCa.

Abstract Background: Glioblastoma (GBM) patients face a dismal prognosis, with median survival of ~15 months and 5-year survival rate of 6.8%. Standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, all GBM patients succumb to their disease. Glioma stem cells (GSCs), a GBM subpopulation, possess enhanced DNA repair that contributes to chemoradiotherapy resistance. The epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM and emerging studies show KDM1A is implicated in DNA damage response. This study aims to determine the significance of KDM1A to promote DNA repair in GSCs. We hypothesize KDM1A inhibition augments therapeutic efficacy of TMZ via attenuation of DNA repair pathways. Methods: Patient-derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination KDM1A knockdown or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR), and non-homologous end-joining (NHEJ) reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ was determined using orthotopic murine GBM models. Results: GBM patient data sets showed KDM1A is elevated in recurrent GBM versus primary tumors. KDM1A knockdown, knockout, or pharmacological inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies showed KDM1A is enriched at the promoters of DNA repair genes. RNA-seq studies confirmed that KDM1A inhibition reduced expression of DNA repair-related genes. RT-qPCR and Western blotting validated downregulation of DNA double-strand break repair genes. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay demonstrated increased TMZ-mediated DNA damage in NCD38-treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of mice bearing orthotopic GBM tumors. Conclusions: Our results provide compelling evidence KDM1A is essential for DNA repair in GSCs and KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination therapy of KDM1A inhibitor NCD38 with TMZ is a potential novel therapeutic strategy to improve GBM outcomes. Citation Format: Salvador Alejo, Bridgitte Palacios, Prabhakar Pitta Venkata, He Yi, Wenjing Li, Jessica Johnson, Sridharan Jayamohan, Weixing Zhao, Siyuan Zheng, Takayoshi Suzuki, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi, Gangadhara R. Sareddy. Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma [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 6263.

Oncoprotein SS18-SSX is a hallmark of synovial sarcomas. However, as a part of the SS18-SSX fusion protein, SS18's function remains unclear. Here, we depict the structures of both human SS18/BRG1 and yeast SNF11/SNF2 subcomplexes. Both subcomplexes assemble into heterodimers that share a similar conformation, suggesting that SNF11 might be a homologue of SS18 in chromatin remodeling complexes. Importantly, our study shows that the self-association of the intrinsically disordered region, QPGY domain, leads to liquid-liquid phase separation (LLPS) of SS18 or SS18-SSX and the subsequent recruitment of BRG1 into phase-separated condensates. Moreover, our results show that the tyrosine residues in the QPGY domain play a decisive role in the LLPS of SS18 or SS18-SSX. Perturbations of either SS18-SSX LLPS or SS18-SSX's binding to BRG1 impair NIH3T3 cell transformation by SS18-SSX. Our data demonstrate that both LLPS and assembling into chromatin remodelers contribute to the oncogenic activity of SS18-SSX in synovial sarcomas.

Endometrial cancer (EC) often exhibit aberrant activation of PI3K/Akt/mTOR signaling and targeted therapies using mTOR inhibitors showed limited success. The epigenetic modifier, lysine-specific histone demethylase-1A (KDM1A/LSD1) is overexpressed in EC, however, the mechanistic and therapeutic implications of KDM1A in EC are poorly understood. Here, using 119 FDA-approved drugs screen, we identified that KDM1A inhibition is highly synergistic with mTOR inhibitors. Combination therapy of KDM1A and mTOR inhibitors potently reduced the cell viability, survival, and migration of EC cells. Mechanistic studies demonstrated that KDM1A inhibition attenuated the activation of mTOR signaling cascade and abolished rapamycin induced feedback activation of Akt. RNA-seq analysis identified that KDM1A inhibition downregulated the expression of genes involved in rapamycin induced activation of Akt, including the mTORC2 complex. Chromatin immunoprecipitation experiments confirmed KDM1A recruitment to the promoter regions of mTORC2 complex genes and that KDM1A inhibition promoted enrichment of repressive H3K9me2 marks at their promoters. Combination therapy of KDM1A inhibitor and rapamycin reduced the tumor growth in EC xenograft and patient derived xenograft models in vivo and patient derived tumor explants ex vivo. Importantly, in silico analysis of TCGA EC patients data sets revealed that KDM1A expression positively correlated with the levels of PI3K/Akt/mTOR genes. Collectively, our results provide compelling evidence that KDM1A inhibition potentiates the activity of mTOR inhibitors by attenuating the feedback activation of Akt survival signaling. Furthermore, the use of concurrent KDM1A and mTOR inhibitors may be an attractive targeted therapy for EC patients.

Demyelination results from the pathological loss of myelin and is a hallmark of many neurodegenerative diseases. Despite the prevalence of demyelinating diseases, there are no disease modifying therapies that prevent the loss of myelin or promote remyelination. This review aims to summarize studies in the field that highlight the importance of nuclear hormone receptors in the promotion and maintenance of myelination and the relevance of nuclear hormone receptors as potential therapeutic targets for demyelinating diseases. These nuclear hormone receptors include the estrogen receptor, progesterone receptor, androgen receptor, vitamin D receptor, thyroid hormone receptor, peroxisome proliferator-activated receptor, liver X receptor, and retinoid X receptor. Pre-clinical studies in well-established animal models of demyelination have shown a prominent role of these nuclear hormone receptors in myelination through their promotion of oligodendrocyte maturation and development. The activation of the nuclear hormone receptors by their ligands also promotes the synthesis of myelin proteins and lipids in mouse models of demyelination. There are limited clinical studies that focus on how the activation of these nuclear hormone receptors could alleviate demyelination in patients with diseases such as multiple sclerosis (MS). However, the completed clinical trials have reported improved clinical outcome in MS patients treated with the ligands of some of these nuclear hormone receptors. Together, the positive results from both clinical and pre-clinical studies point to nuclear hormone receptors as promising therapeutic targets to counter demyelination.

Abstract Background: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis and median survival of 20 months. Standard therapy consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide, and tumor-treating fields. Despite these therapies, GBM patients inevitably experience tumor progression and eventually succumb to their disease. Glioma stem cells (GSCs) play a central role in GBM development and contribute to treatment resistance. Recently we showed that lysine-specific histone demethylase 1A (KDM1A/LSD1) is essential for GSCs stemness, and inhibition of KDM1A induces unfolded protein response (UPR) in GSCs. In this study, we tested the hypothesis that inhibition of KDM1A sensitizes GSCs to ER stress inducers by inducing UPR. Methods: KDM1A knockdown (KDM1A-KD) cells were generated using KDM1A specific shRNA. We studied the effect of KDM1A-KD or pharmacological KDM1A inhibitors (NCD38 and NCL-1) in combination with ER stress inducers (thapsigargin and brefeldin A) on GSCs viability using CellTiter-Glo assay. Stemness was determined using extreme limiting dilution analysis (ELDA) and sphere formation assays. Mechanistic studies were conducted using RNA-seq, ChIP, RT-qPCR, and Western blotting analysis. Furthermore, the in vivo efficacy of KDM1A inhibitor and ER stress inducer was studied using orthotopic models of GBM. Results: Cell viability assays demonstrated that knockdown or inhibition of KDM1A sensitized GSCs to ER stress inducers thapsigargin and brefeldin A. Furthermore, KDM1A-KD, NCD38, or NCL-1, in combination with ER stress inducers, significantly decreased the stemness and sphere-forming ability of GSCs. RNA-seq analysis revealed that UPR was activated after knockdown or inhibition of KDM1A in GSCs. Western blot and RT-qPCR analysis showed that a combination of KDM1A inhibitors and ER stress inducers increased UPR signaling in GSCs. ChIP analysis indicated that KDM1A inhibition enriched the active histone methylation mark (H3K4me2) at the promoter of UPR target genes. In vivo studies showed that KDM1A inhibition activates UPR in tumors and improved overall survival. Conclusions: Our results support that KDM1A knockdown or inhibition sensitizes GSCs to ER stress inducers and that the use of KDM1A inhibitors in conjunction with ER stress inducers is a potential novel therapy for GBM patients. Citation Format: Yi He, Prabhakar Pitta Venkata, Salvador Alejo, Yihong Chen, Bridgitte Palacios, Gabrielle Gray, Uday P. Pratap, Suryavathi Viswanadhapalli, Siyuan Zheng, Rajeshwar R. Tekmal, Andrew J. Brenner, Gangadhara R. Sareddy. KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2026.

Background: Glioblastoma (GBM) is the most common brain tumor. Despite advancements in multimodal treatment strategies, GBM patients exhibit a dismal prognosis with a median survival of 20 months and a 5-year survival rate post diagnosis of 13%. Standard of care consists of surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Despite heavy investment in therapy, GBM patients inevitably experience tumor progression and eventually succumb to their disease. Recently, we showed that lysine-specific histone demethylase 1A (KDM1A), an epigenetic modifier, is overexpressed in GBM. In this study, we tested the hypothesis that KDM1A is essential for DNA damage response (DDR) and inhibition of KDM1A sensitizes GBM to TMZ therapy. Methods: KDM1A knockout (KDM1A-KO) cells were generated using the CRISPR/Cas9 system and knockdown cells were generated using KDM1A specific shRNA (KDM1A-KD). We studied the effect of KDM1A-KO, -KD, or pharmacological KDM1A inhibitors (NCD-38 and NCL-1) on TMZ sensitization using Cell Titer-Glo luminescent cell viability assay, and survival assays. Apoptosis was determined using Caspase 3/7 and TUNEL assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, and Western blot analysis. Furthermore, the in vivo efficacy of KDM1A inhibitor and TMZ therapy was studied using orthotopic models of GBM using both male and female mice. Results: Cell viability and survival assays demonstrated that knockout/knockdown or inhibition of KDM1A sensitized patient derived glioma stem cells (GSCs) to TMZ treatment. Further, combination of NCD-38 or NCL-1 and TMZ significantly increased the apoptosis of GSCs. RNA-seq analysis revealed the modulation of DDR and apoptotic pathways following combination therapy. Pharmacokinetics (PK) and brain bioavailability studies showed that NCD-38 has favorable PK properties and exhibited a significant penetration through the blood brain barrier. Importantly, combination of NCD-38 and TMZ significantly reduced the in vivo tumor progression and improved the overall survival of both male and female mice compared to single drug treatment in GBM orthotopic models. Conclusions: Our results provide evidence that KDM1A knockdown/inhibition sensitizes GBM to TMZ therapy via modulation of DDR and that the use of KDM1A inhibitor in conjunction with TMZ could serve as a novel therapy for GBM patients. Citation Format: Bridgitte E. Palacios, Prabhakar Pitta Venkata, Yihong Chen, Salvador Alejo, Yi He, Suryavathi Viswanadhapalli, Uday Pratap, Junhao Liu, Xiaonan Li, Takayoshi Suzuki, Siyuan Zheng, Andrew Brenner, Ratna Vadlamudi, Gangadhara Sareddy. KDM1A inhibition sensitizes glioblastoma cells to temozolomide therapy through DNA damage and apoptosis pathway modulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6220.

Despite treatment advances, glioblastoma (GBM) is the deadliest tumor of the central nervous system. Patients face a dismal 15-month median survival and average 5-year survival rate of 13%. Current standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, GBM patients often relapse and succumb to their disease. Recently, we have shown that lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM. In this study, we tested the hypothesis that KDM1A is essential for DNA damage response (DDR), inhibiting KDM1A thereby sensitizes GBM to either TMZ or radiation therapy. KDM1A knockout (KDM1A-KO) and knockdown (KDM1A-KD) cells were generated using CRISPR/Cas9 and KDM1A specific shRNA, respectively. Cell viability, clonogenic survival, and apoptotic assays were used to study the effect of KDM1A-KO, -KD, or pharmacological KDM1A inhibitors (NCD-38 and NCL-1) on radiation and TMZ sensitization. Mechanistic studies were conducted using RNA-seq, RT-qPCR, and western blot analysis. Furthermore, in vivo efficacy of KDM1A inhibitor and TMZ was established using orthotopic models of GBM. Cell viability, survival, and apoptotic assays demonstrated that knockout/knockdown or inhibition of KDM1A sensitized GBM cells to both TMZ or radiation therapy. KDM1A inhibitors used in combination with radiation or TMZ significantly increased apoptosis in GBM cells. RNA-seq analysis and signaling studies showed attenuation of DDR pathways in KDM1A-KD cells. Radiation or TMZ treatment enhanced DNA damage in KDM1A-KD cells compared to controls. Importantly, combination of KDM1A inhibitor and TMZ significantly reduced in vivo tumor progression and improved overall survival in orthotopic GBM murine models. Our results provide strong evidence that KDM1A inhibition sensitizes GBM to TMZ or radiation therapy via modulation of DDR. This suggests combination of KDM1A inhibitors with TMZ or radiation therapy could serve as a novel treatment for GBM patients.

Abstract Background: Ovarian cancer (OCa) is the deadliest gynecologic cancer. Recent studies suggest that OCa cells express estrogen receptor beta (ESR2), which functions as a tumor suppressor. However, ESR2 expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms. The lysine-specific histone demethylase 1A (KDM1A), an epigenetic regulator, is overexpressed in OCa. We reason that agents that restore the expression/functions of ESR2 may provide a novel therapeutic opportunity for suppression of OCa. In this study we tested the hypothesis that KDM1A suppresses ESR2 expression/functions and that inhibition of KDM1A potentiates ESR2 mediated tumor suppression. Methods: We examined the utility of combination therapy of KDM1A inhibitor and ESR2 agonist on established and patient derived OCa cells using cell viability, survival, apoptosis and invasion assays. KDM1A knockout (KDM1A-KO) cells were generated using the CRISPR/Cas9 system. Mechanistic studies were conducted using RNA-seq, ERE-Luc reporter assays, RT-qPCR, co-IP, ChIP and Western blot analysis. The efficacy of combination therapy on primary ovarian tumors was examined using patient derived explant (PDEx) models. In vivo efficacy of KDM1A inhibitor and ESR2 agonist was studied using orthotopic OCa and patient derived xenograft models. Results: Analysis of the gene expression profiles of OCa patients showed that KDM1A negatively correlates with ESR2. Combination of KDM1A inhibitor NCD-38 and ESR2 agonist LY500307 synergistically reduced cell viability, survival and invasion and increased apoptosis of established and patient derived primary OCa cells. Inhibition or knockout of KDM1A sensitized OCa cells to ESR2 agonist treatment and significantly increased the expression of ESR2 and its target genes. Further, we found that KDM1A is recruited to the ESR2 0N promoter and interacts with ESR2. In addition, inhibition of KDM1A increased the active histone methylation mark H3K4-me2 at the ESR2 0N promoter. RNA-seq analysis demonstrated that ESR2 agonist treatment resulted in modulation of pathways related to cell cycle, apoptosis and DNA damage. Importantly, combination treatment significantly reduced the tumor growth in orthotopic and patient derived xenograft models and reduced the proliferation of OCa cells in PDEx models. Conclusions: Our results demonstrate that KDM1A inhibition enhances ESR2 expression/functions and combination therapy of KDM1A inhibitor and ESR2 agonist is an attractive therapy for treating OCa. Citation Format: Prabhakar Pitta Venkata, Bridgitte E. Palacios, Yihong Chen, Salvador Alejo, Yi He, Ilanna Loeffel, Uday Pratap, Kristin Altwegg, Suryavathi Viswanadhapalli, Takayoshi Suzuki, Rajeshwar Tekmal, Edward Kost, Gangadhara Sareddy. KDM1A inhibition activates estrogen receptor beta pathway to suppress ovarian cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1764.

Magnetoreceptive animals orient to the earth’s magnetic field at angles that change depending on temporal, spatial, and environmental factors such as season, climate, and position within the geomagnetic field. How magnetic migratory preference changes in response to internal or external stimuli is not understood. We previously found that Caenorhabditis elegans orients to magnetic fields favoring migrations in one of two opposite directions. Here we present new data from our labs together with replication by an independent lab to test how temporal, spatial, and environmental factors influence the unique spatiotemporal trajectory that worms make during magnetotaxis. We found that worms gradually change their average preferred angle of orientation by ~ 180° to the magnetic field during the course of a 90-min assay. Moreover, we found that the wild-type N2 strain prefers to orient towards the left side of a north-facing up, disc-shaped magnet. Lastly, similar to some other behaviors in C. elegans, we found that magnetic orientation may be more robust in dry conditions (< 50% RH). Our findings help explain why C. elegans accumulates with distinct patterns during different periods and in differently shaped magnetic fields. These results provide a tractable system to investigate the behavioral genetic basis of state-dependent magnetic orientation.

PURPOSE:Cancer stem cells (CSCs) are highly tumorigenic, spared by chemotherapy, sustain tumor growth, and are implicated in tumor recurrence after conventional therapies in triple negative breast cancer (TNBC). Lysine-specific histone demethylase 1A (KDM1A) is highly expressed in several human malignancies and CSCs including TNBC. However, the precise mechanistic role of KDM1A in CSC functions and therapeutic utility of KDM1A inhibitor for treating TNBC is poorly understood. METHODS:The effect of KDM1A inhibition on cell viability, apoptosis, and invasion were examined by Cell Titer Glo, Caspase 3/7 Glo, and matrigel invasion assays, respectively. Stemness and self-renewal of CSCs were examined using mammosphere formation and extreme limiting dilution assays. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, Western blotting and reporter gene assays. Mouse xenograft and patient derived xenograft models were used for preclinical evaluation of KDM1A inhibitor. RESULTS:TCGA data sets indicated that KDM1A is highly expressed in TNBC. CSCs express high levels of KDM1A and inhibition of KDM1A reduced the CSCs enrichment in TNBC cells. KDM1A inhibition reduced cell viability, mammosphere formation, self-renewal and promoted apoptosis of CSCs. Mechanistic studies suggested that IL6-JAK-STAT3 and EMT pathways were downregulated in KDM1A knockdown and KDM1A inhibitor treated cells. Importantly, doxycycline inducible knockout of KDM1A reduced tumor progression in orthotopic xenograft models and KDM1A inhibitor NCD38 treatment significantly reduced tumor growth in patient derived xenograft (PDX) models. CONCLUSIONS:Our results establish that KDM1A inhibition mitigates CSCs functions via inhibition of STAT3 and EMT signaling, and KDM1A inhibitor NCD38 may represent a novel class of drug for treating TNBC.

Background: Glioblastomas (GBM) have dismal survival rates (1 year-34.6% and 5 year-4.75%) and affect 13,000 patients yearly. Standard of care treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide (TMZ), and tumor treating fields. Despite heavy investment in therapy, all patients will eventually succumb to their disease. Oncogenic and epigenetic signaling mechanisms may modulate DNA damage response (DDR) in tumors and contribute to chemo and radiation therapy resistance. Recently, we have shown that lysine-specific histone demethylase 1A (KDM1A), an epigenetic regulator, is overexpressed in GBM. In this study, we tested the hypothesis that KDM1A is essential for DDR, and that inhibition of KDM1A induces DNA repair deficiency and sensitizes GBM to TMZ therapy. Methods: To study the role of KDM1A in GBM cells, we have generated KDM1A knockout (KDM1A-KO) cells using the CRISPR/Cas9 system and KDM1A-shRNA transfected primary GBM cells (KDM1A-KD). Effect of KDM1A-KO, -KD or KDM1A inhibitor (NCD-38) on TMZ sensitization was studied using cell viability and survival assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR and western blot analysis. The blood-brain barrier (BBB) permeability of NCD-38 was studied by pharmacokinetic (PK) and brain bioavailability studies. Further, the in vivo efficacy of KDM1A inhibitor was studied using orthotopic models of GBM. Results: Cell viability and survival assays showed that knockout or pharmacological inhibition of KDM1A sensitized GBM cells to TMZ treatment. KDM1A expression is increased in GBM cells after treatment with TMZ. RNA-seq analysis revealed a decreased expression of several genes involved in DNA repair, including MGMT, RAD51, and MRE11A in NCD-38 treated cells compared to control. PK and brain bioavailability studies following a single intravenous dose (IV, 1 mg/kg) and per oral administration (PO, 10 mg/kg) of NCD-38 demonstrated that NCD-38 has favorable PK properties and exhibited a significant penetration of the BBB, with a total brain/plasma ratio of more than two at all tested time points. Further, KDM1A inhibition significantly reduced the in vivo tumor progression in established and primary GBM orthotopic models. Conclusions: Our results provide evidence that KDM1A contributes to chemotherapy resistance in GBM by modulating DNA repair pathways and the use of KDM1A inhibitor in conjunction with standard chemotherapy will serve as novel therapy for GBM patients. Citation Format: Bridgitte E. Palacios, Prabhakar Pitta-Venkata, Yihong Chen, Suryavathi Viswanadhapalli, Uday P. Pratap, Aleksandra Gruslova, Takayoshi Suzuki, Ratna K. Vadlamudi, Andrew Brenner, Gangadhara R. Sareddy. KDM1A inhibition enhances chemotherapy response in glioblastoma via downregulation of DNA repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 273.

Background: Endometrial cancer (EC) is the sixth most common cancer in women. Annually 63,230 new cases are diagnosed with 11,350 deaths estimated in the USA. Currently, advanced EC therapies remain palliative and new therapeutic strategies are urgently needed. Common risk factors include exposure to high levels of estrogen, obesity, and alterations in genetic and epigenetic factors. The lysine-specific demethylase-1A (KDM1A/LSD1) regulates gene expression programs by changing the epigenetic histone marks at the gene promoters. Emerging studies provided the evidence that KDM1A is overexpressed in EC. In studies examining the synthetic lethality of KDM1A inhibition on chemotherapy drug sensitivity, we made an unexpected discovery that KDM1A inhibition potentiate activity of mTOR inhibitors. In this study, we tested the hypothesis that inhibition of KDM1A could sensitize EC to mTOR inhibitor therapy. Methods: To study the significance of KDM1A inhibition on chemotherapy drug sensitivity, we performed MTT assays to screen 119 FDA approved drugs using KDM1A knockdown HEC1A and RL95 EC cell lines. Effect of KDM1A knockdown or KDM1A inhibitor (NCD-38) therapy on EC cells was examined using MTT cell viability assays and clonogenic survival assays. The effect on cell migration was examined using scratch wound healing assay. Mechanistic studies were conducted using RNA-seq, western blot, qRT-PCR, and IHC analysis. The in vivo efficacy of NCD-38 and sirolimus on EC progression was studied using mouse xenograft models. Results: Studies using 119 FDA approved drugs identified that mTOR inhibitors sirolimus and temsirolimus has potent synthetic lethality on KDM1A knockdown cells compared to control cells. Cell viability and survival assays demonstrated that KDM1A knockdown or inhibition in combination with sirolimus synergistically reduced cell viability and the survival of EC cells. Further, combination of KDM1A inhibitor and sirolimus reduced the migration of EC cells. Western blot analysis demonstrated that knockdown or inhibition of KDM1A attenuated the activation of mTOR signaling cascade in EC cells. RNA-seq and gene set enrichment analysis identified the down regulation of E2F pathway and DNA replication pathways in KDM1A and mTOR inhibitor treated cells compared to control cells. Further, combination of NCD-38 and sirolimus significantly reduced the in vivo tumor progression in xenograft models. IHC analysis of tumors revealed the downregulation of proliferation marker Ki67 and phosphorylation of mTOR signaling molecules in combination treated tumors compared to vehicle treated tumors. Conclusions: The results from these studies provide compelling evidence that KDM1A inhibition sensitizes EC cells to mTOR inhibitors, and the use of KDM1A inhibitor in conjunction with mTOR inhibitors may be an attractive therapy for advanced EC patients. Citation Format: Prabhakar Pitta-Venkata, Bridgitte Palacios, Yihong Chen, Suryavathi Viswanadhapalli, Uday P. Pratap, Yiliao Luo, Mengxing Li, Kristin Altwegg, Xiaonan Li, Takayoshi Suzuki, Rajeshwar Rao Tekmal, Edward Kost, Gangadhara Reddy Sareddy. Synthetic lethality of KDM1A and mTOR inhibitors: A novel combination therapy for endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 280.

Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-kappa B (NF-kappa B), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-kappa B target genes, NF-kappa B reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.

ABSTRACTMany animals can orient using the earth’s magnetic field. In a recent study, we performed three distinct behavioral assays providing evidence that the nematode Caenorhabditis elegans orients to earth-strength magnetic fields (Vidal-Gadea et al., 2015). In addition to these behavioral assays, we found that magnetic orientation in C. elegans depends on the AFD sensory neurons and conducted subsequent physiological experiments showing that AFD neurons respond to earth-strength magnetic fields. A new behavioral study by Landler et al. (2017) suggested that C. elegans does not orient to magnetic fields and raises issues that cast doubt on our study. Here we reanalyze Lander et al.’s data to show how they appear to have missed observing positive results, and we highlight differences in experimental methods and interpretations that may explain our different results and conclusions. Moreover, we present new data from our labs together with replication by an independent lab to show how temporal and spatial factors influence the unique spatiotemporal trajectory that worms make during magnetotaxis. Together, these findings provide guidance on how to achieve robust magnetotaxis and reinforce our original finding that C. elegans is a suitable model system to study magnetoreception.