bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 866: Translational repression by a novel partner of human cytoplasmic poly(A) binding protein
Authors: Li Naren , Hu Liang , Liu Qinfang , Xiong Yulan , Yu Jianzhong
Published: June 2022

In higher eukaryotes, post-transcriptional regulation of gene expression, which is accomplished by an ensemble of RNA-binding proteins (RBPs), is critical for cellular homeostasis. Unkempt (UNK) is an evolutionarily conserved Zinc Finger/RING domain RNA-binding protein that was originally identified as a developmental regulator in Drosophila. Recent studies suggest that UNK targets specific mRNAs through its two compact zinc finger clusters and regulates their translation. However, the underlying molecular mechanisms by which UNK represses translation remain unclear. To examine UNK interactions, we purified Flag-tagged UNK and subjected the preparations to Mass spectrometry (MS) and identified an enriched protein Cytoplasmic poly(A)-binding protein(PABPC1), a key component of the translation machinery that binds to the poly(A) tail of mRNAs to promote translation and mRNA turnover. GST Pull down assay and reciprocal immunoprecipitations in HEK293 cells further confirmed the strong interaction between UNK and PABPC1 and showed that the interaction is RNA-dependent. Notably, the structural analysis, along with mutational studies proved that the interaction is mediated by the C-terminal MLLE domain of PABPC1 and the C-terminal Domain of UNK both in vitro and in vivo. Further MS2 tethering assay indicated that UNK inhibits PABPC1-stimulated translation activity. Our studies identified Unkempt as a novel partner of PABPC1 that functions to represses PABPC1-stimulated translation. The findings provide novel insight into the molecular function of Unkempt and PABPC1-mediated translational machinery.

Citation Format: Naren Li, Liang Hu, Qinfang Liu, Yulan Xiong, Jianzhong Yu. Translational repression by a novel partner of human cytoplasmic poly(A) binding protein [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 866.
Abstract 869: Post-transcriptional control of androgen receptor expression
Authors: Likos Eviania , Shukla Girish C.
Published: June 2022

The Androgen Receptor (AR) is a ligand-dependent transcription factor that modulates the expression of androgen-regulated genes involved in the proliferation and differentiation of normal prostate epithelial cells. However, in Prostate Cancer (PCa), these cellular events are dysregulated upon significant changes in AR, such as AR overexpression, leading to uncontrolled cell growth and proliferation. To elucidate the molecular mechanisms by which AR expression and function are controlled, we have explored the post-transcriptional regulatory RNA elements of AR, particularly the 3’ untranslated region (UTR). The AR 3’UTR is 6.8 Kilobases (Kb) long, which indicates that this region is likely a site for multitudinous levels of post-transcriptional regulation. We found that the length of AR 3’UTR negatively correlates with AR expression, with the full length 3’UTR leading to the most downregulation of expression. Currently, we are exploring cis-acting noncoding RNAs and RNA binding proteins (RBPs) predicted to interact with the AR 3’UTR.

Citation Format: Eviania Likos, Girish C. Shukla. Post-transcriptional control of androgen receptor expression [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 869.
Abstract 891: Therapeutic targeting of ovarian cancer stem cells using estrogen receptor beta agonist
Authors: He Yi , Alejo Salvador , Venkata Prabhakar P. , Johnson Jessica D. , Loeffel Ilanna , Martel Julie Ann , Pratap Uday P. , Viswanadhapalli Suryavathi , Tekmal Rajeshwar R. , Vadlamudi Ratna K. , Kost Edward , Sareddy Gangadhara R.
Published: June 2022

Background: Ovarian cancer (OCa) is the deadliest gynecologic cancer. Emerging studies suggest that ovarian cancer stem cells (OCSCs) contribute to tumor relapse and chemotherapy resistance. Recent studies demonstrate that OCa cells express estrogen receptor beta (ERβ), which functions as a tumor suppressor. However, the status of ERβ expression in OCSCs and the therapeutic utility of ERβ agonist LY500307 for targeting OCSCs remain unknown. In this study, we tested the hypothesis that OCSCs express ERβ and that treatment with ERβ agonist reduces stemness and promotes apoptosis of OCSCs.

Methods: We isolated subpopulations of OCSCs from SKOV3, A2780, and patient-derived ascites cells using ALDEFLUOR kit and examined the expression of ERβ in OCSCs using RT-qPCR. The effect of ERβ agonist LY500307 on OCSCs was examined utilizing cell viability, cell cycle, sphere formation, self-renewal, and apoptosis assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, and Western blot analysis. The efficacy of LY500307 on the tumor-initiating capacity of OCSCs was determined via in vivo limiting dilution assay studies using orthotopic intrabursal xenograft murine models.

Results: RT-qPCR assay showed that ERβ, particularly ERβ isoform 1, is highly expressed in OCSCs compared to non-OCSCs. ERβ agonist LY500307 significantly reduced the viability of OCSCs compared to non-OCSCs. Treatment of OCSCs with LY500307 significantly reduced the sphere formation and self-renewal of OCSCs. Further, LY500307 treatment resulted in G2/M cell cycle arrest and promoted the apoptosis of OCSCs. Mechanistic studies using RNA-seq analysis demonstrated that ERβ agonist treatment resulted in the modulation of pathways related to cell cycle and apoptosis. Western blots and RT-qPCR assays confirmed the upregulation of genes involved in apoptosis and cell cycle arrest, such as FDXR and CDKN1A. Importantly, treatment of LY500307 significantly attenuated the tumor-initiating capacity of OCSCs in orthotopically implanted OCa murine models.

Conclusions: Our results demonstrate that ERβ agonist LY500307 is highly efficacious in reducing the stemness and promoting apoptosis of OCSCs and may serve as a novel therapeutic agent in treating OCa

Citation Format: Yi He, Salvador Alejo, Prabhakar P. Venkata, Jessica D. Johnson, Ilanna Loeffel, Julie Ann Martel, Uday P. Pratap, Suryavathi Viswanadhapalli, Rajeshwar R. Tekmal, Ratna K. Vadlamudi, Edward Kost, Gangadhara R. Sareddy. Therapeutic targeting of ovarian cancer stem cells using estrogen receptor beta agonist [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 891.
Abstract 942: Development of customizable bladder cancer mouse model using CRISPR
Authors: Valentine Henkel L. , Satyal Uttam , Abbosh Philip H.
Published: June 2022

Introduction: In forward genetics, a phenotype (sensitivity to therapy) is attributed to a genotype (mutation in relevant gene) based on findings in anecdotal cases (e.g., extreme responders) with little systematic/experimental study. Although powerful, this approach is not widely applicable due to the limited availability of clinical materials and throughput thus limiting the number and type of hypotheses that can be rigorously tested. However, a genotype-therapy sensitivity relationship could be measured in isogenic systems using reverse genetics, whereby mutations in relevant genes are introduced followed by evaluation of a phenotype in wild-type and mutant contexts. Since no such pre-clinical system exists for bladder cancer, we developed an immunocompetent mouse model to induce customizable isogenic tumor formation in bladder urothelium using CRISPR.

Methods: Bladder urothelium was harvested from transgenic mice harboring LSL-Cas9/GFP in Rosa26 locus (as described by Platt et al) and grown as organoids. These organoids had a basal phenotype. AAV harboring Cre and sgRNA(s) were used to induce Cas9 and engineer mutations. Based on their frequent somatic alteration pathway in TCGA-BLCA, Tp53 and Rb1 were chosen for knockout first to establish ‘base organoids’. Additional genes related to PI3K/AKT/mTOR pathway were edited in base organoids to develop a series of organoids with different genotypes predicted to activate this signaling pathway. Sanger sequencing and western blot were used to confirm knockout. Kinase inhibitors targeting the mTOR pathway at different levels were assessed in organoids with different genotypes. The edited urothelial cells were grafted in mouse bladder to develop isogenic tumor to assess in vivo therapeutic vulnerability of in-vitro-prioritized kinase inhibitors matching the genotype of the allografted tumor.

Results: Rb1, Tp53, and Pten were successfully knocked out in urothelial organoids. When treated with Ipataseritib, an AKT inhibitor, the triple knockout organoid was found to be 9X more sensitive than Trp53/Rb1 knockout organoids. In contrast, triple knockout organoids were 15X less sensitive to Gefitinib, an EGFR inhibitor, than Trp53/Rb1 knockout organoids, in line with published data. Edited organoids generate a neo-urothelium in murine bladder in short term assays.

Conclusions: We developed a flexible bladder cancer mouse model to study mutation-therapy relationship using reverse genetics. This system is a plausible way to uncover unappreciated therapeutic vulnerabilities that probably exist in bladder cancer.

Citation Format: Henkel L. Valentine, Uttam Satyal, Philip H. Abbosh. Development of customizable bladder cancer mouse model using CRISPR [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 942.
Abstract 976: Interrogating the pre-metastastic gene signature to block brain metastases
Authors: Kieliszek Agata , Bassey-Archibong Blessing , Venugopal Chitra , Singh Sheila
Published: June 2022

BACKGROUND: The incidence of brain metastases (BM) is tenfold higher than that of primary brain tumors. BM predominantly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need to identify novel therapies against BM. The goal of this work is to uncover the molecular factors that drive the formation of BM and investigate whether we can slow down and ultimately block BM formation.

METHODS: The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from BM patient tumor samples. We use these BM cell lines to generate murine orthotopic xenograft models of BM and interrogate the biological processes that lead to BM. These models have successfully recapitulated all the stages of their respective BM cascades and additionally captured a “pre-metastatic” population of BM cells that have just seeded the brains of mice before forming mature, clinically detectable tumors. Pre-metastatic cell populations are impossible to detect in human patients but represent a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors.

RESULTS: RNA sequencing of pre-metastatic BM cells revealed a unique deregulated transcriptomic profile that is specific to pre-metastatic cells despite the tumor of origin. Subsequent Connectivity Map analysis revealed compounds that we biologically characterized in vitro for selective anti-BMIC phenotypes. This effort led to a lead compound that exhibits anti-BM activity in vitro, while remaining ineffective against normal brain cell controls. Preliminary in vivo work has shown that following both orthotopic and intracardiac injection of BM cells, treatment with this lead compound reduces the tumor burden compared to mice being treated by a vehicle control, while providing a significant survival advantage. Ongoing mechanistic investigations aim to delineate the protein target of this compound in the context of the observed selective anti-BM phenotype.

CONCLUSION: Identification of novel small molecules that target premetastatic BM cells could slow or prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients.

Citation Format: Agata Kieliszek, Blessing Bassey-Archibong, Chitra Venugopal, Sheila Singh. Interrogating the pre-metastastic gene signature to block brain metastases [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 976.
Abstract 98: The role of HORMAD1 in transformation of keratinocytes following vemurafenib treatment
Authors: Gantchev Jennifer , Villarreal Amelia Martinez , Ramchatesingh Brandon , Litvinov Ivan V.
Published: June 2022

BRAF inhibitor (BRAFi) therapies are highly effective treatments for metastatic melanoma. However, BRAFi monotherapies are associated with a significant risk of squamous cell carcinoma (SCC) development in melanoma patients. Consequently, BRAF inhibition results in an unanticipated activation of the MAPK pathway in surrounding cells that harbour wildtype BRAF. Fortunately, a combination approach with both a BRAFi and the selective MAPK inhibitor (MEKi), trametinib, attenuates BRAFi-induced risk of SCCs. Despite the fact that this combination therapy is widely used in clinical practice, the mechanisms associated with SCC induction and why this effect is attenuated with a MEK inhibitor remain poorly understood. HORMAD1, a meiosis specific gene, has been shown to have an impact on genomic instability and proliferation in the development of SCCs. Moreover, recent work in our lab has demonstrated that the MAPK pathway regulates HORMAD1 expression in SCC cell lines. Therefore, we sought to investigate a potential role of HORMAD1 in the risk and development of SCCs as a mechanism of BRAFi that is attenuated with the co-administration of the MEKi, trametinib. Using the BRAFi, vemurafenib, we inhibited the kinase activity of activated BRAF (V600E) and the MEKi, trametinib to evaluate the expression of HORMAD1 in melanoma and normal keratinocyte cell lines. To determine the effects of these two inhibitors, a cell proliferation assay was conducted followed by the evaluation of various components of cell cycle regulation and genomic instability. We found that HORMAD1 protein expression levels increased following treatment with the BRAFi, vemurafenib, in the normal keratinocyte cell line, HaCaT, but not in the melanoma cell line, A375. In contrast, HORMAD1 expression decreased following treatment with the MEKi, trametinib in both HaCaT and A375. Cell proliferation assays depict a vulnerability to both vemurafenib and trametinib in both normal and melanoma cell lines, however, HaCaT cells appear to be more susceptible to increased levels of genomic instability following treatment with vemurafenib when compared to A375. Our results suggest that HORMAD1 likely plays a key role in the development of SCCs by increasing genomic instability and proliferation in normal keratinocytes following BRAFi monotreatment. The increased risk of SCC formation is attenuated by dampening HORMAD1 levels with the co-administration of the MEKi, trametinib.

Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in transformation of keratinocytes following vemurafenib treatment [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 98.
Abstract 988: Epigenetic upregulation of neuropilin-1 promotes osteosarcoma progression and metastasis
Authors: Nerlakanti Niveditha , Mcguire Jeremy , Yu Diana , Reed Damon , Lynch Conor C.
Published: June 2022

Background: Osteosarcoma (OS) frequently metastasizes to lung and the prognosis for these patients is grim with 5-year overall survival rates as low as 30%. There has been little change in the treatment paradigm for this disease and new targeted therapies are an urgent and unmet clinical need. Previously, we showed that the epigenetic regulators, histone deacetylase 1 and 2 (HDAC1 and 2) are key mediators of OS progression and lung metastasis and that the HDAC1/2 selective inhibitor (romidepsin) significantly prevented OS lung metastasis in vivo. Yet, inhibition of HDACs by romidepsin has noted toxicities in humans and here we define the potential mechanisms through which HDAC1/2 mediate their effects in a bid to identify additional therapeutic targets.

Methods: Human OS cell lines were treated with vehicle or romidepsin for 24h before undergoing RNA/protein isolation for microarray/proteomics to identify differentially regulated genes. Standard siRNA, proliferation, migration and molecular analyses were preformed to investigate the role of candidate genes in OS malignant behavior.

Results: Microarray/proteomics analyses on romidepsin treated OS cells identified neuropilin-1 (NRP1), a membrane bound co-receptor for vascular endothelial growth factor, to be highly downregulated in response to romidepsin; (<70% compared to vehicle in multiple OS cell lines). NRP1 knockdown (siRNA) led to reduced cell proliferation and interestingly, increased cell migration. Further, a phosphokinase array revealed increased phosphorylation in eNOS, ERK 1/2 and β-catenin in NRP1 knockdown cells that is consistent with a role for NRP1 in regulating OS migration. Based on this data, we hypothesize that NRP1 is a significant regulator of OS proliferation and migration. Our next steps will be to dissect the role of NRP1 silencing in OS invasion and metastasisin vivo and the potential role of NRP1 in OS differentiation.

Conclusions: HDAC1/2 contributes to OS growth potentially via the upregulation of NRP1, a therapeutically actionable target.

Citation Format: Niveditha Nerlakanti, Jeremy Mcguire, Diana Yu, Damon Reed, Conor C. Lynch. Epigenetic upregulation of neuropilin-1 promotes osteosarcoma progression and metastasis [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 988.
Abstract CT245: First in human CD200 activation receptor ligand and tumor lysate vaccine immunotherapy for recurrent glioblastoma in adults
Authors: Olin Michael R. , Neil Elizabeth C. , Eaton Anne , Lunn Shannon , Moertel Christopher L.
Published: June 2022

Background: Immunotherapy has revolutionized clinical management of malignancies rendering cancers that previously had a dismal prognosis more manageable. Unfortunately, these therapies can cause serious immune-related adverse events, including death. In CNS cancers, currently available immunotherapies have limited to no efficacy. Our work in glioblastoma (GBM), which is considered one of the most aggressive cancers as well as being universally fatal, is focused on the CD200 immune checkpoint that modulates the immune system through the inhibitory receptor (CD200R1) and activation receptor (CD200AR). The peptide checkpoint ligand (CD200AR-L) was developed to target the CD200AR that downregulates the inhibitory PD-1/PD-L1, CTLA4 and CD200R immune checkpoints, permitting immune system activation that is less hampered by the inhibitory effects of GBM. Following a positive clinical trial in dogs that spontaneously developed high-grade gliomas, we initiated a first-in-human Phase 1 Study of the CD200 Activation Receptor Ligand (CD200AR-L) and Allogeneic Tumor Lysate Vaccine (GBM6-AD) immunotherapy for Recurrent Glioblastoma (NCT04642937).

Methods: This is a single-center, first-in-human, dose-escalation Phase 1 clinical trial utilizing a 3+3 design at three dose levels; 3.75micrograms/kg, 5micrograms/kg, and 7.25micrograms/kg. Adults 18 years and old with recurrent GBM and no pre-exiting immune-related condition plus adequate performance score were eligible. Accrual started in December 2020 at dose level 1 with a second cohort having received treatment on dose level 2. CD200AR-L is administered on days 1 and 2 by suprascapular intradermal injection following the application of topical imiquimod. On day 2, a fixed dose of the allogeneic vaccine is injected intradermally at the same site. The “Induction Phase” consists of this injection series weekly for 4 weeks with monitoring for dose-limiting toxicity through Day28. The “Maintenance Phase” doses the vaccine series monthly for 2 months, then the “Booster Phase” doses every eight weeks for 24 months or until end of treatment. Patients are monitored for adverse events, including immune-mediated intracranial edema and other immune-related complications, and their neurological function is scored per NANO criteria. Correlative studies include immune monitoring (including expression of CD200R1, PD1/PD-L1, and CTLA-4) and MRI imaging assessment per iRANO criteria.

Citation Format: Michael R. Olin, Elizabeth C. Neil, Anne Eaton, Shannon Lunn, Christopher L. Moertel. First in human CD200 activation receptor ligand and tumor lysate vaccine immunotherapy for recurrent glioblastoma in adults [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 CT245.
Abstract CT534: Magnesium treatment on the demethylation of chemokine (C-X-C motif) ligand 9 (CXCL9) gene, results from the personalized prevention of colorectal cancer trial
Authors: Zhu Xiangzhu , Huang Xiang , Zheng Yinan , Zhang Wei , Shu Lihua , Ness Reid , Murff Harvey J , Yu Chang , Shrubsole Martha J , Hou Lifang , Dai Qi
Published: June 2022

Background: Chemokine ligand 9 (CXCL9), a T-cell chemoattractant, plays a critical role in regulating antitumor immunity in cancer immunotherapy, i.e., clinical responses to anti-PD(L)-1 treatment. Very recently, CXCL9 was identified as the strongest contributor to the “inflammatory clock of aging”, linked to age-related chronic inflammation. Due to the critical role of magnesium (Mg) in regulating inflammatory responses, this study aims to investigate the effect of Mg treatment on DNA methylation changes in CXCL9.

Methods: The Personalized Prevention of Colorectal Cancer Trial conducted at Vanderbilt University Medical Center was a double-blind 2 × 2 factorial randomized controlled trial of 240 participants who completed the study. Participants aged from 40 to 85 years, had a daily calcium intake 700-2000 mg and calcium:magnesium intake ratio ≥2.6 based on the average of the two baseline 24-hour dietary recalls.

Results: Among three CpG sites, a 12-week personalized dose of Mg supplementation marginally increased the 5-hydroxymethylcytosine methylation, a measure of active demethylation, at CpG site 04038163 (3'UTR of CXCL9 gene) by 7.0% compared to the placebo group (p=0.07). Although the overall effect was not significant or of borderline significance, Mg treatment significantly reduced the levels of 5-hydroxymethylcytosine by 3.7% at the CpG site 12793812 (body of CXCL9 gene) among those with aged 60 years or above (p=0.03).

Conclusions: These findings indicate that Mg treatment modified the demethylation process in the CXCL9 gene, a critical regulator of immune responses and inflammation. Future studies are needed to examine if Mg treatment also changes the circulating levels of CXCL9.

Citation Format: Xiangzhu Zhu, Xiang Huang, Yinan Zheng, Wei Zhang, Lihua Shu, Reid Ness, Harvey J Murff, Chang Yu, Martha J Shrubsole, Lifang Hou, Qi Dai. Magnesium treatment on the demethylation of chemokine (C-X-C motif) ligand 9 (CXCL9) gene, results from the personalized prevention of colorectal cancer trial [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 CT534.
Abstract LB039: VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition
Authors: Pilie Patrick G. , Zhou Lijun , Zhang Yan-Ting , Peterson Christine , McGrail Daniel , Peng Yang , Peng Guang , Liu Xiande , Zhang Xuesong , Jonasch Eric
Published: June 2022

Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability, but lacks mutations in canonical DNA damage response (DDR) genes. Bi-allelic loss of VHL is a near ubiquitous, truncal event in ccRCC development. Herein, we performed preclinical and clinical analysis to understand how VHL loss impacts DDR pathway activity and how specific DDR deficiencies (DDR-D) may be targeted in ccRCC.

Methods: We performed DNA whole exome sequencing (WES) and reverse phase protein array (RPPA) early stage ccRCC tumors. All patients were consented under an IRB-approved protocol for the use of tissue. Transcriptomic and protein analyses of select DDR pathways was performed on in silico data (TCGA). DDR functional assays and ATR inhibitor treatment were performed on preclinical cell line models, and in vivo ATR inhibitor treatment in ccRCC xenografts with varying VHL status.

Results: Bi-allelic loss of VHL via mutation in one allele and copy number loss of chromosome 3p was observed in 13/13 patient tumors. Mutations in other genes associated with ccRCC and/or canonical DDR genes were not observed. Despite VHL loss being the only driver mutation, these early stage tumors displayed tumor mutational burden (TMB) similar to ccRCC across all tumor stages (1.1 ± 0.5 mut/Mb). Proteomic analysis of patient tumor versus normal tissue (N=12) revealed significantly different (P<0.05) mean expression level of 218 proteins, including significantly lower phospho-ATM at the autophosphorylated S1981 site and lower phospho-CHK2 at the downstream T68 site. VHL-/- cells display defective binding to and repressed activation of Tip60 (KAT5), a key acetyltransferase that activates ATM kinase and aids in the selection of DNA repair pathways following damage. Lastly, treatment with ATRi (VE-822) of 786-O and 786-VHL overexpressing tumor xenografts showed ATRi was safe and significantly inhibited tumor growth preferentially in the 786-O VHL null model.

Conclusions: Here we show in patient tissue and preclinical models that VHL biallelic loss results in genomic instability and functional impairment of ATM kinase activity; furthermore, we provide preclinical evidence for selectively targeting the ATR signaling cascade in patients with VHL-deficient kidney cancer. Clinical trials of ATR inhibitor based therapy in patients with advanced cancer, including ccRCC, are underway (e.g. NCT04266912).

Citation Format: Patrick G. Pilie, Lijun Zhou, Yan-Ting Zhang, Christine Peterson, Daniel McGrail, Yang Peng, Guang Peng, Xiande Liu, Xuesong Zhang, Eric Jonasch. VHL-deficient renal cell carcinoma displays defective ATM activation and sensitivity to ATR inhibition [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 LB039.
Abstract LB089: Effective in vivo treatment of endometrial tumor models with coexistent mutant PI3K and PTEN inactivation with a selective bi-steric mTORC1 kinase inhibitor
Authors: Solomon Hilla , Mukherjee Radha , Chen Xiaoping , Zhao HuiYong , de Stanchina Elisa , Weigelt Britta , Schram Alison M. , Aghajanian Carol , Singh Mallika , Smith Jan , Rosen Neal
Published: June 2022

Dysregulation of PI3K signaling is a common event in human cancer, but feedback inhibition of receptor kinase signaling and/or induction of PTEN translation by activated PI3K/AKT/mTOR signaling reduces pathway output and tumor dependency. In some cancers, including the majority of endometrial carcinomas, these lesions coexist and cause synergistic hyperactivation of pathway output, particularly mTORC1 kinase signaling. Although this suggests that PI3K signaling is a driver of these endometrial cancers, standard inhibitors have had only marginal activity in patients with advanced disease. We here show that, in experimental isogenic models and PDX models with PI3K and PTEN alterations, PI3K inhibitors and rapalogs are weak inhibitors of TORC1 targets and slow but do not suppress the in vivo growth of these tumors. Revolution Medicines has developed a series of bi-steric selective and potent mTORC1 inhibitors that inhibit the phosphorylation of 4EBP-1, S6K and other mTORC1 substrates at concentrations that do not affect TORC2 substrates and thus do not induce the hyperglycemia caused by downregulation of AKT signaling by pan-mTOR kinase inhibitors. We have explored the biologic and anti-tumor effects of these inhibitors in models of endometrial cancer with coexistent PI3K mutation and PTEN inactivation. The mTORC1 kinase selective bi-steric inhibitor RMC-6272 can potently suppress both mTORC1 activity and the growth of these tumor cells in vitro. Moreover, in contrast to PI3K inhibition, RMC-6272 effectively inhibited the phosphorylation of mTORC1 kinase substrates and completely suppressed the growth in vivo of multiple PDXs with this genotype without inducing hyperglycemia or causing weight loss in the mice. The results suggest that dysregulation of TORC1 activity is an important driver of endometrial carcinomas with coexistent PTEN inactivation and PI3K mutations and that selective TORC1 kinase inhibitors may prove to be useful therapeutics in this context. The bi-steric mTORC1 kinase inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952).

Citation Format: Hilla Solomon, Radha Mukherjee, Xiaoping Chen, HuiYong Zhao, Elisa de Stanchina, Britta Weigelt, Alison M. Schram, Carol Aghajanian, Mallika Singh, Jan Smith, Neal Rosen. Effective in vivo treatment of endometrial tumor models with coexistent mutant PI3K and PTEN inactivation with a selective bi-steric mTORC1 kinase inhibitor [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 LB089.
Abstract LB098: Integrative approaches to modulate antigen presentation and boost cancer immune response
Authors: Gu Shengqing , Zhang Wubing , Wang Xiaoqing , Jiang Peng , Freeman Gordon J. , Rodig Scott J. , Long Henry , Gewurz Benjamin E. , Hodi F. Stephen , Liu X. Shirley , Brown Myles
Published: June 2022

Immune checkpoint blockade (ICB) therapy revolutionized cancer treatment, but patients with impaired MHC-I and/or MHC-II expression show inferior response. We observed differential expression patterns for MHC-I and MHC-II in cancer cells and applied multiple approaches to examine their regulatory mechanisms. To identify the modulators of MHC-I, we combined FACS-based genome-wide CRISPR screens with data-mining from public data. We identified TRAF3, a suppressor of the NFkB pathway, as a negative regulator of MHC-I. The Traf3-knockout gene expression signature is associated with better survival in ICB-naïve patients with cancer and better ICB response. We then screened for drugs with similar transcriptional effects as this signature and identified Second Mitochondria-derived Activator of Caspase (SMAC) mimetics. We experimentally validated that the SMAC mimetic birinapant upregulates MHC-I, sensitizes cancer cells to T cell-dependent killing, and adds to ICB efficacy. In contrast to MHC-I, the expression of MHC-II shows dramatic intra- and inter-sample heterogeneity. To identify the regulators of MHC-II, we conducted data-mining of the transcriptomic and proteomic data from Cancer Cell Line Encyclopedia (CCLE) and multiple melanoma clinical cohorts. We found that a higher cancer-cell-intrinsic MHC-II level is significantly associated with better anti-PD-1 response, and that the Hippo signaling pathway can regulate MHC-II expression in melanoma cells. Our findings provide preclinical rationales for increasing cancer cell MHC-I/MHC-II expression to enhance sensitivity to immunotherapy.

Citation Format: Shengqing Gu, Wubing Zhang, Xiaoqing Wang, Peng Jiang, Gordon J. Freeman, Scott J. Rodig, Henry Long, Benjamin E. Gewurz, F. Stephen Hodi, X. Shirley Liu, Myles Brown. Integrative approaches to modulate antigen presentation and boost cancer immune response [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 LB098.
Abstract LB116: CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma
Authors: Walle Thomas , Kraske Joscha A. , Liao Boyu , Timke Carmen , Lenoir Benedicte , Halama Niels , Huber Peter E.
Published: June 2022

The role of radiotherapy in the treatment of pancreatic adenocarcinoma (PDAC) is controversial. In many other tumor types, radiotherapy is a mainstay therapy whose anti-tumor effects have partially been ascribed to T cell responses. The role of Natural Killer (NK) cells in radiotherapy tumor responses remains unclear. Here, we focused on the role of NK cells in radiotherapy of pancreatic cancer.

In a randomized controlled pancreatic cancer trial, we found that CXCL8 serum levels increased under radiotherapy and NK cells positively correlated with prolonged overall patient survival. Accordingly, NK cells preferentially infiltrated irradiated tumors and exhibited CD56dim like cytotoxic transcriptomic states. In preclinical experimental systems in mice and tumor cells, NF-κB and mTOR signaling prompted radiation-induced CXCL8 secretion from senescent tumor cells causing directional migration of CD56dim NK cells into tumors. Thus, we link senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy which may be integrated into PDAC treatment algorithms.

Citation Format: Thomas Walle, Joscha A. Kraske, Boyu Liao, Carmen Timke, Benedicte Lenoir, Niels Halama, Peter E. Huber. CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma [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 LB116.
Abstract LB130: High-throughput RNA sequencing directly from cell lysates enables reproducible phenotypic profiling for assessing novel compounds and treatments in cancer research
Authors: O'Hara Andrea , Stephens Michael , DeVito Ilaria , Turner Laure , Mozdzierz Christopher , Latif Haythem , Zhou Ginger
Published: June 2022

New oncology therapies are driven by multiple approaches, with a heavy emphasis on small-molecule therapeutics to target cancer cells and provide a therapeutic benefit to cancer patients. These small-molecule compounds can be used as a stand-alone treatment approach or may be combined with other compounds to bring enhanced effects. Compound therapeutic discovery relies heavily on high-throughput screening assays for measurement of phenotypic responses following compound treatment of oncology cell cultures which can range from very targeted assays to wide-ranging unbiased approaches. Standard whole transcriptome RNA-seq is a preferred method for unbiased measurement of phenotypic responses but is often overlooked due to limited scalability and high sample screening costs.

To address these challenges, we have developed a high-throughput gene expression (HT-GEx) assay that combines a variety of strategies to enable a low-cost and high-throughput alternative to standard RNA-seq. First, a simplified workflow removes upstream RNA isolation steps and tags transcripts directly from cell lysate. This is achieved by incorporating both a sample barcode and a unique molecular index (UMI) during the reverse transcription reaction. Next, we leverage a 3’ end counting approach to enable a reduction in sequencing depth coverage (i.e. sample cost) without compromising gene detection sensitivity. With these key advances, a high-plex and high-throughput screening assay is achievable at a reduced cost by removing the need to purify RNA, tagging transcripts early in the workflow to allow pooling of samples and reducing sequencing depth. We have combined these high-throughput and cost-saving strategies and present results which confirm HT-GEx offers results on par with standard RNA-seq. Gene detection sensitivity based on number of genes detected per millions of reads is highly similar, with gene detection sensitivity saturated at approximately 2 million reads. Additionally, the number of genes detected between replicates for RNA samples and lysate samples demonstrate strong linear correlation, implying highly reproducible results. Thus, high-throughput gene expression is an ideal method for phenotypic screening in oncology cell lines following stand-alone or combined compound treatments.

Citation Format: Andrea O'Hara, Michael Stephens, Ilaria DeVito, Laure Turner, Christopher Mozdzierz, Haythem Latif, Ginger Zhou. High-throughput RNA sequencing directly from cell lysates enables reproducible phenotypic profiling for assessing novel compounds and treatments in cancer research [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 LB130.
Abstract LB159: Neighborhood obesogenic environment and risk of prostate cancer: The Multiethnic Cohort
Authors: Hebert-DeRouen Mindy C. , Tao Li , Shariff-Marco Salma , Yan Juan , Shvetsov Yurii B. , Park Song-Yi , Albright Cheryl L. , Monroe Kristine , Marchand Loic Le , Wilkens Lynne , Gomez Scarlett L. , Cheng Iona
Published: June 2022

Background: Obesity is associated with risk of aggressive prostate cancer. It is not known whether neighborhood obesogenic factors are associated with prostate cancer risk.

Methods: Neighborhood socioeconomic status (nSES) and four neighborhood obesogenic environment factors (urbanicity, mixed-land development, unhealthy food environment, and parks) were assessed for associations with prostate cancer risk among 41,563 African American, European American, Japanese American, and Latino males in the Multiethnic Cohort Study (MEC), California site. Multivariable Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for non-aggressive and aggressive prostate cancer, adjusting for individual-level sociodemographic, behavioral, and prostate cancer risk factors. Analyses were stratified by race, ethnicity, and, among Latino males, nativity.

Results: Males residing in low SES, compared to high SES, neighborhoods had lower risk of non-aggressive prostate cancer (lowest vs. highest quintile HR=0.81; 95% CI=0.68, 0.95, p-trend 0.024); driven by a similar trend among U.S.-born and foreign-born Latino males. Foreign-born Latino males in neighborhoods with low mixed-land development had increased risk (lowest vs. highest quintile HR=1.52; 95% CI=1.09, 2.12). African American males in neighborhoods with more unhealthy food environments had increased risk of aggressive disease (more vs. none, HR=1.35; 95% CI=1.02, 1.77).

Conclusions: Neighborhood SES and obesogenic factors were independently associated with prostate cancer risk; associations varied by race, ethnicity, nativity, and disease aggressiveness.

Impact: Upstream structural and social determinants of health that contribute to neighborhood obesogenic characteristics likely impact prostate cancer risk differently across groups defined by race, ethnicity, and nativity and by disease aggressiveness.

Citation Format: Mindy C. Hebert-DeRouen, Li Tao, Salma Shariff-Marco, Juan Yan, Yurii B. Shvetsov, Song-Yi Park, Cheryl L. Albright, Kristine Monroe, Loic Le Marchand, Lynne Wilkens, Scarlett L. Gomez, Iona Cheng. Neighborhood obesogenic environment and risk of prostate cancer: The Multiethnic Cohort [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 LB159.
Abstract LB506: Structure based small molecule drug design for targeting WNK1 in triple negative breast cancer
Authors: Kannangara Ashari Rashmi , Rodriguez Melanie , Chlebowicz Julita , Akella Radha , Cobb Melanie H. , Tambar Uttam , Goldsmith Elizabeth J.
Published: June 2022

Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors lacking estrogen, progesterone, and HER-2 receptors. It accounts for 10-20% of breast cancers, appears in younger women, metastasizes fast, and is associated with poor prognosis and poor patient survival. There are no available tailored therapies for TNBC to date. One of the ways to treat TNBC is by targeting its metastasis feature. With-no-lysine kinase 1 (WNK1), which is known to interact with diverse signaling pathways including ion homeostasis, has been recently identified as one of 6 driver genes in a murine TNBC model. Published and preliminary data support the premise that WNK1 promotes epithelial-mesenchymal transition (EMT) and invasion in TNBC. Genetic and pharmacological inhibition of WNK1 blocks metastasis in a TNBC mouse model. Given the existence of four WNK isoforms in which kinase domains are 90% identical, developing a clinically available WNK1-specific inhibitor is an unmet need. We identified WNK1-selective inhibitors by a High Throughput Screen (HTS) of over 200,000 structurally diverse small molecules. A class of quinoline-derived compounds showed high specificity and potency for WNK1. A combination of molecular modeling, crystallography, and synthesis-based strategies were employed to improve the potency of compounds. Interestingly, the compound SC107-01 reported below 100 nM IC50 for WNK1 by in vitro assays. Also, it shows higher specificity for WNK1 over other kinases as evident by kinome-specificity profiling. We had previously crystallized the kinase domain of WNK1. The structure revealed its unique active site. Co-crystal structure of SC107-01 with WNK1 shows that the inhibitor binds to the WNKs’ specific active site and forms a unique set of interactions. Further modifications to the SC107-01 are underway to improve bioavailability and efficacy. In vivo toxicology and drug pharmacokinetics will be conducted in mouse models. We anticipate that this work will lay the foundation for developing a small molecule that inhibits WNK1 to combat metastasis in TNBC.

Citation Format: Ashari Rashmi Kannangara, Melanie Rodriguez, Julita Chlebowicz, Radha Akella, Melanie H. Cobb, Uttam Tambar, Elizabeth J. Goldsmith. Structure based small molecule drug design for targeting WNK1 in triple negative breast cancer [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 LB506.
Abstract LB513: In vivo efficacy screen of a CSF-1R inhibitor ABSK021 in mouse syngeneic tumor models reveals immune features that may predict its therapeutic response
Authors: Zhu Zhixuan , Peng Juan , Shi Wan , Chen Zhui , Yang Shuqun
Published: June 2022

Colony-stimulating factor 1 (CSF-1) and its receptor CSF-1R play critical roles in immune suppressive and tumor promoting characteristics of tumor-associated macrophages (TAM). ABSK021 is a highly potent and selective inhibitor of CSF-1R that is currently under investigation in phase 1b clinical trials of cancer patients. However, it remains to be elucidated what population might be sensitive to CSF-1R inhibitor. Syngeneic mouse models with complete and functional immune system are important for the translation of preclinical immuno-oncology research into clinical settings. Here, we performed an efficacy screen in ten syngeneic models to evaluate the response to CSF-1R inhibition by ASBK021, and profiled the transcriptomic features by RNA-sequencing in each model. In vivo efficacy studies showed that four models were sensitive to CSF-1R inhibition (tumor growth inhibition value (TGI) ≥ 35%) while six models were insensitive (TGI < 35%). Differentially expressed gene (DEG) analysis and gene set enrichment analysis (GSEA) revealed that the MHC class II genes were down-regulated and immune-related pathways had lower enrichment level in the sensitive models, when compared with insensitive models. Further, the result of in silico immune cell deconvolution by CIBERSORT indicated that the activated CD8+ T cells had significantly negative correlation with TGI in this screen. Taken together, these observations suggested the predictive value of certain immune activation features for the sensitivity to the inhibition of CSF-1R signal.

Citation Format: Zhixuan Zhu, Juan Peng, Wan Shi, Zhui Chen, Shuqun Yang. In vivo efficacy screen of a CSF-1R inhibitor ABSK021 in mouse syngeneic tumor models reveals immune features that may predict its therapeutic response [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 LB513.
Abstract LB526: HBI-2375, a selective inhibitor of MLL1-WDR5 interaction, possesses desirable preclinical characteristics to be pursued in IND enabling studies and future clinical investigations
Authors: Shojaei Farbod , Semple J E. , Fang Che , Ricono Jill M. , Kabbinavar Fairooz , Goodenow Bob , Gillings Mireille
Published: June 2022

92130MLL1 (mixed lineage leukemia1) interaction with WDR5 (WD repeat domain 5) is critical for MLL1 histone methyl transferase (H3K4me) activity leading to transcriptional regulation of gene expression. MLL1 dysregulation such as its translocation at chromosome 11q23 results in generation of MLL1 fusion genes and leukemia development. Additionally, recent studies indicated a role for MLL1-WDR5 interaction in solid tumors progression via directly affecting cancer cells or by suppression of tumor immunity. Therefore, MLL1-WDR5 interaction is an important target in cancer drug discovery, both in leukemia and in solid tumors. HUYABIO recently completed acquisition of a novel series of MLL1-WDR5 inhibitors and conducted an additional SAR campaign to identify a lead compound with greater efficacy and PK (pharmacokinetics)characteristics. Characterization of a series of analogs identified HBI-2375 as a selective inhibitor of MLL1-WDR5 interaction. TR-FRET peptide binding assay indicated potent binding of HBI-2375 to WDR5 (IC50=4.48 nM) and in vitro cell potency studies showed inhibition of proliferation in MV4;11 leukemia cells by HBI-2375 (IC50=3.17 µM).Furthermore, HBI-2375 was stable in liver S9 and whole blood and displayed reasonable PK properties (Cmax, T1/2, AUC and F%) in preclinical testing in mouse and dog. In vivo efficacy studies in MV4;11 model showed 86% and 77% tumor growth inhibition in HBI-2375 treated tumors when dosed at 80 mpk or 40 mpk (po, qd x 21) respectively. In addition, HBI-2375 was found to be well tolerated since body weight data in the recipients did not show a significant change throughout the treatment. Interestingly, ex vivo tumor analysis showed a reduction in H3K4me in both 40 and 80 mpk samples, further validating target-driven activity of HBI-2375. These data resulted in the nomination of HBI-2375 as the lead clinical candidate for the current IND enabling studies and future investigations in the clinic.

Citation Format: Farbod Shojaei, J E. Semple, Che Fang, Jill M. Ricono, Fairooz Kabbinavar, Bob Goodenow, Mireille Gillings. HBI-2375, a selective inhibitor of MLL1-WDR5 interaction, possesses desirable preclinical characteristics to be pursued in IND enabling studies and future clinical investigations [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 LB526.