bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6368: Discovery of potent, orally bioavailable, SOS1 inhibitors for KRAS-driven tumors
Authors: Sasmal Sanjita , Patra Sukanya , Boorgu Venkatesham , Yanamadra Mahesh , Ettam Ashok , Dunaboyina Nagaraju , Kummari Githavani , Yengala Ram Mohan , Lakhavath Balakrishna , Das Jayita , Sunkanapally Satheesh , Reddy Pravalika , Mathew Megha Mariam
Published: June 2022

KRAS is an oncogene implicated in a wide variety of tumors (~21% of solid tumors harbor KRAS mutations). KRAS interaction with Guanidine Exchange Factors (GEFs) is crucial for its activation, with SOS1 being the predominant GEF. SOS1 inhibition is thus expected to be an effective strategy for targeting the downstream signaling pathway, resulting in anti-proliferative activity in RAS-driven cancers. We have identified multiple potent and selective SOS1 inhibitors, demonstrating significant reduction of GEF activity in a dose-dependent manner. The lead compounds show anti-proliferative activity across a panel of WT and mutant KRAS cell lines, and are synergistic with MAPK pathway inhibitors including KRASG12C inhibitor Sotorasib. Correspondingly, significant reduction in PD biomarkers, pERK and pAKT is demonstrated in KRAS mutant cell lines. PK-PD correlation is also established in a tumor bearing mice model, with dose-dependent reduction of both pERK and pAKT. The lead compound shows good ADME properties, and is orally bioavailable, making it amenable for further in vivo profiling.

Citation Format: Sanjita Sasmal, Sukanya Patra, Venkatesham Boorgu, Mahesh Yanamadra, Ashok Ettam, Nagaraju Dunaboyina, Githavani Kummari, Ram Mohan Yengala, Balakrishna Lakhavath, Jayita Das, Satheesh Sunkanapally, Pravalika Reddy, Megha Mariam Mathew. Discovery of potent, orally bioavailable, SOS1 inhibitors for KRAS-driven tumors [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 6368.
Abstract 6369: Discovery of potent, orally bioavailable, brain penetrant RAD51 inhibitor as an anti-cancer agent
Authors: Sasmal Sanjita , Patra Sukanya , Boorgu Venkatesham , Chithaluri Shankar , Yanamadra Mahesh , Hiremath Deepika , Pathange Hemashankar , Girme Sandeep M. , Pawar Amitkumar , Shinde Rutuja Narayan , Samanta Nilanjan
Published: June 2022

Proliferating cells undergo DNA damage, and this is more pronounced in cancer cells due to their high rate of proliferation. Cancer cells are thus more dependent on the DNA damage repair pathway for their survival. Double-strand breaks (DSBs) are amongst the most severe type of DNA damage and are predominantly repaired by homologous recombination (HR) in an error-free manner. RAD51 is a pivotal recombinase for DSB repair by the HR pathway. Binding of RAD51 with BRCA2 followed by its nuclear translocation, is one of the key repair mechanisms for RAD51 mediated DSB repair. We have identified novel and potent RAD51 inhibitors disrupting the RAD51:BRCA2 interaction, which can address BRCA2 WT patient population. Upon induction of exogenous DNA damage, our compounds inhibit nuclear RAD51 foci formation and demonstrate sustained γH2AX accumulation in the nucleus, suggesting persistent DNA damage. Our orally bioavailable & brain penetrant lead compound shows anti-proliferative activity in multiple BRCA2 WT cancer cell lines across various indications, and excellent synergy when combined with PARP1 inhibitors such as Olaparib. Further, in a wound healing assay, treatment with our lead compound inhibits cell migration in a dose-dependent manner. TNBC patients with extensive brain metastasis have limited treatment options. Thus, a brain penetrant RAD51 inhibitor could provide a novel treatment option in this setting.

Citation Format: Sanjita Sasmal, Sukanya Patra, Venkatesham Boorgu, Shankar Chithaluri, Mahesh Yanamadra, Deepika Hiremath, Hemashankar Pathange, Sandeep M. Girme, Amitkumar Pawar, Rutuja Narayan Shinde, Nilanjan Samanta. Discovery of potent, orally bioavailable, brain penetrant RAD51 inhibitor as an anti-cancer agent [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 6369.
Abstract 6372: Nanodrug delivery system for non-genotoxic p53 activator INZ-C
Authors: Bhattarai Nimisha , Wang Jieqiong , Nguyen Daniel , Yang Xiaoxiao , Helmers Linh , Paruch Jennifer , Li Li , Zhang Yiwei , Meng Kun , Wang Alun , Jayawickramarajah Janarthanan , Wang Binghe , Zeng Shelya , Lu Hua
Published: June 2022

p53 is a known tumor suppressor protein whose gene is altered in 50% of all human cancers, while its function is inactivated in the many cancers harboring wild type p53, making it an attractive target for targeted cancer therapies. In this regard, our lab had previously discovered a small molecule, Inauhzin (INZ C), as a potent non-genotoxic p53 activator. In an effort to improve the bioavailability and efficacy, we have now encapsulated the INZ C small molecule in a chitosan/cyclodextrin nanoparticle delivery system. Evaluation of in vitro/in vivo activity and protien expression suggests nanoparticle encapsulation of INZ-C (n-INZ-C) improved p53 induction and inhibition of cell growth in lung and colorectal cancers, while maintaining a minimal effect for MEF and Wi38 normal cells. These results are consistent with the significantly reduced cellular uptake observed for normal cells as compared to the cancer cells. H/E staining of tissues for mice treated with n-INZ-C reveals no toxicity to other organs as well. Additionally, n-INZ-C demonstrates the ability to block migration of cells in vitro as demonstrated by both wound healing and trans-well migration assays. Our in vivo pharmacokinetic evaluation suggests nanoparticle encapsulation doubles the half-life of INZ-C from 2.5 to 5 h. These results suggest that nanoparticle encapsulation improved activity and bioavailability of INZ-C while maintaining the non-toxic nature of INZ-C in normal cells. In addition, we unveiled GRP78 as a potential new target of INZ-C and found out that knockdown of GRP78 can alleviate the inhibitory effect of INZ-C on lung and colorectal cancer proliferation. Further studies now focus on revealing the mechanism of enhanced cellular uptake for INZ-C in cancer cells as comapred to normal cells. Thus, these results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic and also identified a novel target of INZ-C.

Citation Format: Nimisha Bhattarai, Jieqiong Wang, Daniel Nguyen, Xiaoxiao Yang, Linh Helmers, Jennifer Paruch, Li Li, Yiwei Zhang, Kun Meng, Alun Wang, Janarthanan Jayawickramarajah, Binghe Wang, Shelya Zeng, Hua Lu. Nanodrug delivery system for non-genotoxic p53 activator INZ-C [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 6372.
Abstract 6397: Understanding the glioblastoma microenvironment with spatial resolution in PDX models
Authors: Abdelkareem Aly O. , Osz Katalin , Senger Donna , Chan Jennifer A. , Morrissy Sorana
Published: June 2022

Glioblastoma multiforme (GBM) is the most common adult brain tumour, and despite aggressive treatment, it recurs fatally. GBM tumours include diverse populations of malignant and non-neoplastic cells with distinct molecular capabilities and with differential levels of sensitivity to treatment. Understanding the cell dynamics that occur during the development of GBM resistance to therapy could reveal key aspects of this process, including how resistance is acquired in time and how the diverse cell types of the tumour microenvironment (TME) contribute to this phenotype. In addition to the role of the TME, GBM exhibits significant tumour heterogeneity with diverse genetic clones coexisting in the same tumor, as well as cells with similar genetic backgrounds capable of adopting distinct transcriptional states and subtypes. The complex and dynamic interactions between tumor and TME remain to be fully studied. This work focuses on the in vivo spatial organization in GBM during disease progression. We generated spatial transcriptomic data from a set of adult GBM samples grown as patient-derived xenograft (PDX) models, profiled at different time points of the disease. Three PDX lines from one GBM patient (derived from tumor core, vascularized area, and infiltrating front) were used to recapitulate the genetic and phenotypic heterogeneity observed in the human disease. Two replicates from each of 8 PDX mice were collected from early, mid, and late time points of tumour growth and data was generated using the 10X Genomics Visium platform. We developed a robust computational pipeline capable of distinguishing admixture of human (tumour) and mouse cells (TME), using state-of-the-art tools. Human and mouse cell types and states were identified using pooled and separate single-cell references of human GBM states, and mouse brain cells from both normal and tumour conditions. With this approach we observe spatially distinct patterns of both (a) tumour infiltration patterns specific to the each PDX line that includes non-random distribution of GBM transcriptional states and genetic clones, and (b) spatially distinct infiltration of TME components including microglial and macrophage populations. Overall, our approach addresses the challenge of understanding the tumor-TME relationship by application of spatial profiling in PDX models, and provides a computational pipeline complex multi-species analysis in the spatial transcriptomic field.

Citation Format: Aly O. Abdelkareem, Katalin Osz, Donna Senger, Jennifer A. Chan, Sorana Morrissy. Understanding the glioblastoma microenvironment with spatial resolution in PDX models [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 6397.
Abstract 6399: SP in relation to NK1R and Ki67 predicts survival in hormone receptor negative breast cancer
Authors: Al-Keilani Maha S. , Elstaty Rana , Alqudah Mohammad A.
Published: June 2022

Background: Substance P (SP) is a tachykinin that has been implicated in carcinogenesis in several types of cancer through binding to neurokinin 1 receptor (NK1R). This study aimed to investigate the expression levels of SP and NK1R in hormone receptor negative (HR -ve) versus positive (HR +ve) breast cancer tissues and their relationship with clinicopathological characteristics of patients, Ki-67 index, and overall survival rate.

Methods: Immunohistochemical expression levels of SP, NK1R and Ki-67 were measured in 144 paraffin-embedded breast cancer tissues (77 HR -ve and 67 HR +ve). SP and NK1R were scored semiquantitatively, while Ki-67 was obtained by the percentage of total number of tumor cells with nuclear staining. The optimal cutoff value for SP, NK1R and Ki-67 were assessed by Cutoff Finder. Pearson Chi-square (χ2) and Fisher’s exact tests were used to compare the staining scores between groups. ANOVA and student’s t-test were used to compare group means. Kaplan-Meier method with log-rank test was used for survival analysis.

Results: High expression of SP, NK1R and Ki-67 was observed in 41%, 36.8% and 38.9% of cases, respectively. Association analyses revealed that SP expression in HR -ve breast cancer was higher in pN3 tumors (p=0.002), positive axillary lymph node metastasis (p=0.003), and high NK1R expression (p=0.010). In HR+ve breast cancer, SP was lower in HER2 positive tumors (p=0.001). Regarding NK1R, the association analysis showed that low NK1R expression level in HR +ve tumors was related to stage IV (p=0.023), pN1 (p=0.006), and low Ki-67 index (p=0.038). Survival analysis revealed that high SP level negatively impacted overall survival in HR-ve tumors that had low NK1R level (p=0.021). Moreover, high SP negatively impacted overall survival in HR-ve tumors that had low Ki-67 level (p=0.005). Nevertheless, NK1R expression did not impact survival in HR-ve or HR+ve cases despite stratification by SP and Ki-67 expression (p>0.05).

Conclusion: Up to our knowledge, this is the first study to investigate the prognostic potential of SP and NK1R in breast cancer. Combined expression levels of SP and NK1R or Ki-67 could be used to predict survival in breast cancer patients with HR-ve tumors. Our findings suggest the potential role of SP and NK1R as therapeutic targets in breast cancer.

Citation Format: Maha S. Al-Keilani, Rana Elstaty, Mohammad A. Alqudah. SP in relation to NK1R and Ki67 predicts survival in hormone receptor 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 6399.
Abstract 65: A novel tool for functional characterization of mutations and their response to targeted therapy
Authors: Barbash Zohar , Haham DIkla , Fillipov-Levy Natalie , Cohen Limor , Birnbaum Lea , Rotkopf Shay , Kifer Ilona , Tarcic Gabi
Published: June 2022

Genomic profiling of tumors has uncovered staggering amounts of novel alterations, termed variants of uncertain significance (VUS), even in well-studied oncogenes. Strikingly, as the number of sequenced samples increases so does the number of VUS. While the study of alterations identified as VUS has shown that some are inconsequential, it has also been shown that many can cause protein activation and are involved in oncogenicity and resistance to therapy or might serve as novel drug targets. We developed a high-throughput cell-based assay that allows functional annotation of VUS and their sensitivity to different inhibitors. The system quantifies signaling pathway activation using fluorescent imaging coupled with image analysis of cells expressing the mutated protein together with a fluorescently labeled signaling pathway reporter. We show that this method accurately identifies known driver mutations in 10 different oncogenes, including receptor tyrosine kinases and signaling proteins, spanning the four major signaling pathways involved in cancer. We then deployed this system to characterize over 100 different VUS in these genes. We also show that the method correctly recapitulates differential sensitivity of cKIT and EGFR mutations to different inhibitors. Finally, we highlight the importance of concurrent mutations in KRAS which abrogate the response to EGFR inhibitors. Altogether, this system provides significant information both for development of new targeted agents as well as tailoring of optimal treatment for patients.

Citation Format: Zohar Barbash, DIkla Haham, Natalie Fillipov-Levy, Limor Cohen, Lea Birnbaum, Shay Rotkopf, Ilona Kifer, Gabi Tarcic. A novel tool for functional characterization of mutations and their response to targeted therapy [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 65.
Abstract 657: Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung cancer
Authors: Isozaki Hideko , Abbasi Ammal , Nikpour Naveed , Stanzione Marcello , Sakhtemani Ramin , Monroe Susanna L. , Shaw Alice T. , Lin Jessica J. , Sequist Lecia V. , Piotrowska Zofia , Buisson Rémi , Lawrence Michael S. , Hata Aaron N.
Published: June 2022

Acquired drug resistance to even the most effective anti cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We have seen that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug tolerant cancer cells persisting during therapy. Preventing therapy induced A3A mutagenesis by gene deletion delayed the emergence of drug resistance. Here, we show that therapy induced A3A mutagenesis contributes to tumor evolution in NSCLC. Whole genome sequencing revealed that resistant clones that evolved from persistent drug tolerant cells (late clones) harbored more A3A mutations compared to the parental cell population than pre existing resistant clones (early clones). In a subset of NSCLC patients who received targeted therapies, we observed A3A mutations accompanied clonal evolution during treatment. Comparison of APOBEC mutation fractions in short vs long term responders suggests that short responders with acquired resistance mechanisms that evolved from pre existing resistant clones have less accumulation of APOBEC mutations. Collectively, these findings insist that an increase in mutagenic processes drives tumor evolution during targeted therapy treatment and leads to acquired resistance. Thus, suppressing expression or enzymatic activity of A3A may represent a potential therapeutic strategy to halt the evolution of resistant clones and prevent acquired resistance to lung cancer targeted therapy.

Citation Format: Hideko Isozaki, Ammal Abbasi, Naveed Nikpour, Marcello Stanzione, Ramin Sakhtemani, Susanna L. Monroe, Alice T. Shaw, Jessica J. Lin, Lecia V. Sequist, Zofia Piotrowska, Rémi Buisson, Michael S. Lawrence, Aaron N. Hata. Impact of therapy induced APOBEC3A mutagenesis on tumor evolution in non small cell lung 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 657.
Abstract 661: Androgen deprivation therapy allows for effective anti-TIGIT immunotherapy in murine model of castration resistant prostate cancer
Authors: Polesso Fanny , Guan Xiangnan , Xia Zheng , Moran Amy E.
Published: June 2022

Prostate cancer is the second leading cause of cancer associated mortality in men, and for reasons that remain unclear, immunotherapy treatments have largely failed in patients with advanced prostate cancer. Standard of care therapy for these patients relies on androgen deprivation therapies (ADT) to block testosterone production combined with small molecules that inhibit androgen synthesis and/or the function of the androgen receptor (AR). Importantly, androgens are reported to be immunosuppressive; therefore, we postulated that androgen axis blockade might promote response to immunotherapy. In the recent years, the co-inhibitory receptor TIGIT has emerged as a promising immunotherapy target, and we observed that TIGIT is highly expressed on tumor infiltrating T cells and NK cells in metastatic castration resistant prostate cancer (CRPC) patients treated with the AR antagonist enzalutamide. Likewise, in a mouse model of CRPC, we show that tumor infiltrating T cells express TIGIT and that treatment with enzalutamide increases its expression. In this model, neither treatment with anti-TIGIT alone nor with chemical castration and enzalutamide affects tumor growth. Remarkably, the combination of anti-TIGIT with chemical castration and enzalutamide leads to delayed tumor growth and long-term cures in about 60% of the animals. Taken together, our data suggest that androgen receptor blockade sensitizes immune cells in the tumor to respond to anti-TIGIT immunotherapy.

Citation Format: Fanny Polesso, Xiangnan Guan, Zheng Xia, Amy E. Moran. Androgen deprivation therapy allows for effective anti-TIGIT immunotherapy in murine model of castration resistant prostate 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 661.
Abstract 671: Cryo-electronmicroscopy of PI3K reveals additional regulatory features
Authors: Hart Jonathan R. , Yang Su , Liu Xiao , Xu Yingna , Zou Xinyu , Zhang Huibing , Zhou Qingtong , Xia Tian , Zhang Yan , Yang Dehua , Wang Ming-Wei , Vogt Peter K.
Published: June 2022

Many solid tumors have alterations in phosphoinositide 3-kinase (PI3K) signaling. These can occur through mutation of the p110alpha and p85alpha proteins of PI3Kalpha itself as well as through alterations to upstream receptor tyrosine kinases (RTKs) and phosphatases of PIP3, including PTEN. As a whole, PI3K signaling is one of the most frequently altered in cancer. These mutations lead to activation of the PI3K complex and to constitutive downstream signaling. This signaling allows cells to enter a pro-growth and pro-survival state which is beneficial to the cancer cells.

The structure of PI3K has been extensively studied by crystallography and NMR. Here, we present our work on the full-length complex of p110alpha with p85alpha using cryo-EM. When compared with the previous structural studies, cryo-EM shows a similar structure and major structural features are unchanged. However, our cryo-EM studies revealed several additional structural features of the PI3K complex.

Cryo-EM showed that SH3, BH and cSH2 domains of p85alpha can bind to p110alpha. When inhibited with Alpelisib, these domains form a stable cover of the active site of the kinase. These domains interact with the helical and kinase domains of p110alpha and the iSH2 domain of p85alpha. The position of these domains restricts access to the kinase active site and may contribute to the inhibition of PI3K activity by Alpelisib.

When the PI3K complex binds to phosphorylated RTKs, the nSH2 binds to phosphotyrosine and relieves inhibitory interactions with the helical domain of p110alpha. Using cryo-EM, we have studied this activation by incubating the full-length PI3K complex with synthetic phosphopeptides. 2D cryo-EM class averages showed a dramatic rearrangement of the PI3K complex. In addition to the expected displacement of the nSH2 domain, the ABD and iSH2 domains undergo structural changes.

Acknowledgments: This work was supported by the National Cancer Institute of the National Institutes of Health under awards R35 CA197582 (PKV) and R50 CA243899 (JRH).

Citation Format: Jonathan R. Hart, Su Yang, Xiao Liu, Yingna Xu, Xinyu Zou, Huibing Zhang, Qingtong Zhou, Tian Xia, Yan Zhang, Dehua Yang, Ming-Wei Wang, Peter K. Vogt. Cryo-electronmicroscopy of PI3K reveals additional regulatory features [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 671.
Abstract 679: Inhibition of Notch signaling causes progenitor-to-hepatocyte differentiation and regressions in a subset of hepatocellular carcinoma by controlling C/EBPα expression and HNF4α activity
Authors: Seidel Kerstin , Piskol Robert , Nguyen Thi Thu Thao , Cottonham Charisa , Merchant Mark , Siebel Christian
Published: June 2022

​​Notch signaling regulates cell fate decisions during development and maintenance of many organs. During hepatogenesis, an active NOTCH2 signal is required for specification of the cholangiocyte lineage, while hepatoblasts remaining in a Notch-off state contribute to the default hepatocyte lineage. Notch pathway activation has been reported in liver tumors, although Notch-related liver cancer mutations are rare. We identified a subset of hepatocellular carcinoma that is exceptionally sensitive to the inhibition of a JAG1-NOTCH2 signal in a mouse preclinical trial using 47 liver cancer PDX models.

Transcriptional profiling revealed that the sensitive models resemble a progenitor-like state. Highly selective therapeutic antibodies targeting either the JAG1 ligand or the NOTCH2 receptor triggered hepatocyte differentiation programs and tumor regressions.Single cell analyses revealed heterogeneous transcriptional states at baseline, some of which were profoundly perturbed upon Notch inhibition. Topic modeling combined with inference of transcription factor activities suggested the involvement of HNF4α and C/EBPα in treatment induced changes. Through its target HES1, Notch signaling directly represses C/EBPα expression. Notch inhibition derepresses C/EBPα which then binds chromatin in cooperation with HNF4α. We found that this cooperative binding results in expression of genes associated with mature hepatocyte activity, which was incompatible with tumor maintenance.

Importantly, we identify hallmarks to aid the identification of this relatively rare tumor type. We suggest that inducing cell states towards differentiated, less aggressive states, especially using targeted single-agent therapeutics, holds great promise for the treatment of certain tumors in the liver and, by extension, possibly in other progenitor-like cancers.

Citation Format: Kerstin Seidel, Robert Piskol, Thi Thu Thao Nguyen, Charisa Cottonham, Mark Merchant, Christian Siebel. Inhibition of Notch signaling causes progenitor-to-hepatocyte differentiation and regressions in a subset of hepatocellular carcinoma by controlling C/EBPα expression and HNF4α activity [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 679.
Abstract 681: Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition
Authors: Olsen Sarah Naomi , Godfrey Laura , Healy James P. , Hatton Charles , Armstrong Scott A.
Published: June 2022

MLL1 (KMT2A) translocations lead to aberrant expression of stem cell associated gene programs in hematopoietic cells producing a particularly aggressive subtype of leukemia, namely MLL-rearranged acute myeloid and lymphoblastic leukemia (AML and ALL, respectively). In pediatric leukemia, the most common rearrangement is the t(9; 11) (p22; q23) reciprocal translocation, which results in the expression of the MLL-AF9 fusion gene. Since the MLL-AF9 rearrangement does not lead to direct activation of an enzyme, small molecule drug discovery remains challenging and despite decades of research on MLL-rearranged leukemia, we have been limited by the inability of acutely assessing the consequences of direct MLL-AF9 inactivation in relevant model systems. By coupling rapid PROTAC-mediated degradation of the oncogenic MLL-AF9 fusion protein with genome-wide gene expression and chromatin analyses we have now been able to identify the immediate transcriptional and chromatin state consequences of MLL-AF9 degradation. Specifically, we have established a core set of MLL-AF9 target genes whose expression changes within minutes of MLL-AF9 degradation. These rapid expression changes are mediated by changes in productive RNA Polymerase II elongation and increased RNA Polymerase II pausing. At later timepoints, degradation of the fusion protein also induces loss of an active chromatin landscape at MLL-AF9 target genes, characterized by loss of MLL-AF9 associated proteins and activating histone modifications. These insights improve our mechanistic understanding of how MLL-AF9 mediates chromatin remodeling and leukemogenesis, while also helping us evaluate small molecule inhibitors of epigenetic mechanisms that target the MLL-AF9 protein complex. Specifically, we investigated how degradation of MLL-AF9 compares to enzymatic inhibition of DOT1L or inhibition of the MENIN-MLL interaction. Interestingly, both DOT1L and MENIN inhibitors fell short when comparing their effects to MLL-AF9 degradation and only combined DOT1L/MENIN inhibition induced global MLL-AF9 destabilization on chromatin, thus disrupting the full MLL-AF9 directed epigenetic and transcriptional program. Ultimately, we were able to confirm these findings in a patient-derived xenograft model in vivo, where only the combination was able to significantly prolong leukemia free survival. In summary, our studies establish the cellular and molecular consequences of targeted degradation of the MLL-AF9 fusion oncoprotein and help improve our understanding of small molecule inhibitors of epigenetic mechanisms as cancer therapies.

Citation Format: Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Charles Hatton, Scott A. Armstrong. Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin 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 681.
Abstract 691: Antineoplastic effect of marina crystal minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo
Authors: El-Din Nariman K. Badr , El-Dein Mai Alaa , Ghoneum Mamdooh
Published: June 2022

Purpose: Marina crystal minerals (MCM) is a crystallized mixture of minerals and trace elements from sea water. Earlier in vitro studies have shown that MCM possesses apoptotic and immune modulatory effects in human breast cancer cells MDA-MB-231. The current study aimed to evaluate the anticancer effect of MCM in vivo against the growth of murine mammary adenocarcinoma cells and to explore the mechanisms underlying its effect.

Materials and Methods: Mice were inoculated intramuscularly with Ehrlich ascites carcinoma (EAC) cells, a breast adenocarcinoma of mouse origin. The tumors grew and became palpable within 9 days post-inoculation. Tumor-bearing mice were injected with MCM intraperitoneally (IP) or intratumorally (IT) at a dose of 40 mg/kg BW for 6 days/week. Different parameters were evaluated, including tumor growth, cell cycle progression, cell cycle regulatory proteins, apoptosis, apoptotic regulatory markers, mitochondrial membrane potential (MMP), natural killer (NK) cell activity, and histopathological effects.

Results: Treatment with MCM caused a reduction in tumor volume by 49.4% for IP and 59.5% for IT injection. MCM induced apoptosis in cancer cells, as indicated by the appearance of a sub-G1 peak and confirmed by Annexin V/PI assay and histopathological examination. This was mediated by increased Bax expression, caspase-3 activation, decreased Bcl-2 expression, and MMP disruption. Furthermore, MCM treatment induced G1 cell cycle arrest which was mediated through significantly increased expression of p53, p21, and p27 and decreased expression of Cyclin D1 and PCNA in cancer cells. Finally, MCM treatment markedly enhanced NK cell cytotoxicity.

Conclusion: MCM possesses chemopreventive potential to reduce tumor growth by suppressing cell proliferation, inducing apoptosis in EAC cells via a mitochondrial dependent pathway, and activating the immune system. Our results suggest that MCM may have clinical implications for the treatment of breast adenocarcinoma in humans. MCM was provided by the Foundation for Basic Research Institute of Oncology, Japan.

Citation Format: Nariman K. Badr El-Din, Mai Alaa El-Dein, Mamdooh Ghoneum. Antineoplastic effect of marina crystal minerals (MCM) against the growth of murine mammary adenocarcinoma cells in vivo [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 691.
Abstract 712: Cytokine and TGF-β levels are associated with changes in cervicovaginal microbiota in a cohort of Caribbean women
Authors: Rodríguez Eduardo Lemuel Tosado , Dominicci-Maura Anelisse , Mendez Loyda , Dorta Stephanie , Romaguera Josefina , Godoy-Vitorino Filipa
Published: June 2022

Recent studies suggest that the cervical microbiome can strongly influence inflammation and pre-cancerous lesion progression. However, research focused on understanding the role of microbial communities in the progression of pre-cancerous lesions to cancer in Latino women is scarce. We aimed to investigate the relationship between the cervicovaginal microbiome and inflammation while considering cervical neoplasia and HPV infection in Puerto Rican women. We collected cervical swabs and lavages from 142 participants coming to colposcopy clinics in San Juan, Puerto Rico. Genomic DNA was extracted from swabs, and 16S rDNA V4 region genes were amplified and sequenced by Illumina MiSeq. Inflammatory (IL-1β, TNFa, IFNg, IL-6), anti-inflammatory (IL-4, IL-10, TGFβ1), and trafficking (IL-8, MIP1a, MCP1, IP10) cytokines were measured from cervical lavages, using Luminex MAGPIX technology. Cytokines were related to microbes via an inflammation scoring index based on the quartile and tercile distribution of the cytokine’s concentration. IL-10 (p value= 0.0455) was significantly different when evaluating HPV risk, while IL1-β (p value= 0.0005) and INF-γ (p value= 0.0258) were significant when evaluating cervical disease. We found significant differences in diversity and composition of the microbiota among HPV risk, cervical disease, pro-inflammatory, anti-inflammatory, and trafficking cytokine abundances. The increasing concentration of IL1-β, IL-10, and INF-γ, associated with a decrease in Lactobacillus communities. While contrarily, bacteria associated with dysbiosis such as Gardnerella, Prevotella, Atopobium increased. This study also revealed that the most dominant community state types (CST) among Puerto Rican women regardless of lesion or HPV status, are CST3 and 4 featuring high diversity and anaerobic bacteria typical of vaginosis in Caucasians. These CSTs are especially abundant with ~ 90% dominance in participants with high grade disease (HGSIL) and high-risk HPV. Our study evidence that the cervical microbiota of Puerto Ricans is characteristically diverse and that the joint host-microbe interaction analyses via cytokine signaling and microbiota in pre-cancerous lesions has great translational potential.

Citation Format: Eduardo Lemuel Tosado Rodríguez, Anelisse Dominicci-Maura, Loyda Mendez, Stephanie Dorta, Josefina Romaguera, Filipa Godoy-Vitorino. Cytokine and TGF-β levels are associated with changes in cervicovaginal microbiota in a cohort of Caribbean women [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 712.
Abstract 765: Hybridized-capture sequencing methods bias analysis of antigen presentation genes
Authors: Medina Egmidio , Campbell Katie , Ribas Antoni
Published: June 2022

Introduction: Tumor-immune dynamics are often interrogated using hybridized capture-based sequencing approaches which require the use of baits that target the protein-coding regions of the genome. However, the genes encoding the antigen presentation machinery, including human leukocyte antigen (HLA) proteins, are very polymorphic, resulting in thousands of unique genotypes across patient cohorts. Here, using whole exome sequencing (WES) and hybridized capture-based RNA sequencing (RNAseq) from melanoma patients, we show that these genes are not uniformly assessed by common sequencing methods, and require careful analysis for accurate detection of copy number and somatic alterations.

Methods: WES and RNAseq data was analyzed from 760 melanoma tumor biopsies, with WES derived from patient-matched normal blood samples. Coverage metrics for 26 antigen presentation genes, including classical and non-classical HLA genes, were evaluated, and the difference in coverage was calculated for heterozygous alleles. Each individual was assessed for heterozygous single nucleotide polymorphisms (SNPs) within each allele, and with respect to the haplotype of the human reference genome.

Results: Focusing on the 26 genes assessed, HLA-A, HLA-B, HLA-C, and HLA-DRB1 were the most diverse across our patient cohort; there was an average of 134 unique pairs of HLA alleles in these genes. We compared the SNP profiles of allele combinations to the haplotype of the human reference genome, and observed four patterns: heterozygous alleles are similar to each other and the reference haplotype, heterozygous alleles are different from each other and different to the reference, alleles are very similar to each other and greatly differ from the reference, or one allele is more similar to the reference haplotype. The fourth pattern showed the greatest bias in sequencing, where the allele more similar to the human reference genome had a median 4-fold increase in coverage in normal WES data.

Conclusions: Capture-based sequencing shows selection bias, as exome probes have been designed based upon the reported human reference genome, but results in selection bias in hybridized capture sequencing approaches. Alleles that have higher polymorphic differences with respect to the reference are not equally captured, resulting in nonuniform sequencing of heterozygous alleles.

Citation Format: Egmidio Medina, Katie Campbell, Antoni Ribas. Hybridized-capture sequencing methods bias analysis of antigen presentation genes [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 765.
Abstract 772: Single cell whole genome sequencing to assess tumor heterogeneity in endometrial cancer
Authors: Villarreal Enrique Velazquez , Gibbs Lee D. , Mahinbakht Dana , Da Silva Diane M. , Roman Lynda D. , Craig David W. , Carpten John D.
Published: June 2022

Introduction: Endometrial cancer (EC) is the most common gynecological malignancy among women in developed countries. In the United States, it is estimated that 66,570 new cases and 12,940 deaths will occur in 2021. EC is more common in white women, but black women have a worse prognosis with higher mortality rates. Molecular characteristics in tumor tissue have been reported for potential clinical utility. Integrated genomic characterization efforts have shown common molecular alterations occurring in oncogenic pathways from bulk molecular profiling of EC tumors. However, little is known about the tumor heterogeneity in endometrial cancer. Thus, using high resolution single cell technologies, we assessed tumor heterogeneity in endometrial cancers. Our preliminary study includes endometrial tumors from five (3 African Americans, 1 Hispanic/Latino, 1 Caucasian) cases. We utilize bulk whole exome sequencing and single cell whole genome sequencing copy number variation to study tumor heterogeneity. Frozen tumor sections were used to collect and sequence single nuclei utilizing the 10x ChromiumTM single cell copy number variation (scCNV). Illumina NovasEQ600 was used to sequence the single cell libraries.

Results: 1,546 cells were sequenced across five tumors. Each tumor was visually inspected through hierarchical clustering from the scCNV data. Beyond describing the heterogeneity through chromosome or chromosome arm level gains and losses in samples that included single and mixed histology tumors, we assessed clonal populations suggesting apparently different clonal evolution within samples. Mutations in cancer related genes such as TP53, PTEN and PIK3CA among others suggested a clonal tumor cell stratification. The single cell whole genome data was compared to bulk exome sequencing data to determine the power of scCNV to detect clonal populations of tumor cells.

Conclusions: Using varied histologies in five EC tumors, we performed DNA-CNV analysis using single cell sequencing. This level of resolution enhanced our analysis to provide clear evidence of heterogeneity in all tumors that were assessed in our study. We plan to further expand our sample size to study the degree of heterogeneity in endometrial cancer.

Citation Format: Enrique Velazquez Villarreal, Lee D. Gibbs, Dana Mahinbakht, Diane M. Da Silva, Lynda D. Roman, David W. Craig, John D. Carpten. Single cell whole genome sequencing to assess tumor heterogeneity in endometrial 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 772.
Abstract 773: Inhibition of ubiquitin receptor ADRM1 for the treatment of metastatic breast cancer
Authors: Andrews Christopher , Yates Clayton , Karanam Balasubramanyam
Published: June 2022

Adhesion-regulating molecule 1 (ADRM1) is a polyubiquitin receptor on the 26S proteasome. ADRM1 is upregulated in many cancers. In this study, we evaluated the potential prognostic and predictive value of ADRM1 in breast cancer patients. We determined the expression of ADRM1 and its relationship to breast cancer subtypes, using the Cancer Genome Atlas dataset that contained racial and clinical outcomes data totaling 1108 patients. The ADRM1 mRNA expression levels were divided into quartiles to designate the ADRM1 high versus low expression designation. Karanam et. al developed an orally available proteasome inhibitor bis-benzylidine piperidone (RA190), that binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory particle of the proteasome and directly kills cancer cells by triggering proteotoxic stress. RA190 binds to cysteine 88 of ADRM1/RPN13, triggering rapid accumulation of high-molecular-weight poly-ubiquitinated proteins resulting in apoptosis. Up284 is one of a second generation of small molecules that mimic RA190 activity. The objective of this study is to verify the role of ubiquitin receptor RPN13/ADRM1 in metastatic breast cancer samples of European American and African American patients and identify the safe combination of regimens of bortezomib in combination with Up289 to mitigate recurrence, metastasis and reduce mortality in AA TNBC/QNBC. Clinically, this will reveal a new, hitherto unknown targeted regimen for AA QNBC. From the biological perspective, it will provide insights into the underlying molecular changes that are associated with health disparities. Currently available treatments are unable to eradicate metastatic breast cancer specifically those classified as QNBC. Our preliminary analysis of Cancer Genome Atlas (TCGA) indicated that ADRM1 expression is highly expressed in AA versus EA breast cancer patients, resulting in a worse overall survival. Further analysis of ADRM1 in QNBC patients indicated that it was significantly elevated predominantly in African American patients with QNBC.

Citation Format: Christopher Andrews, Clayton Yates, Balasubramanyam Karanam. Inhibition of ubiquitin receptor ADRM1 for the treatment of metastatic 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 773.
Abstract 782: Multiomic single cell sequencing identified regulatory elements in prostate cells
Authors: Tian Yijun , Soupir Alex , Wang Liang
Published: June 2022

Introduction: Despite significant progress, the genetic role of regulatory elements in gene expression is still poorly understood in prostate cells. Recent development in single cell sequencing by combining ATAC-seq and RNA-seq has made it possible to determine genome-wide linkages between chromatin accessibilities and gene expression.

Methods: To test the feasibility of using single cell multiome sequencing in dissecting regulatory linkages between chromatin accessibilities and gene expressions, we applied 10X Genomics Chromium Single Cell Multiome ATAC + Gene Expression platform to simultaneously encapsulate Tn5 transposase tagged nuclei from two prostate cell lines. We applied down-sampling linkage analysis to determine highly reproducible genome-wide associations between ATAC-seq peaks and RNA-seq gene expressions. To investigate the chromatin accessibilities related to human transcription factor binding sites, we scanned the JASPAR motif collection with genome-wide Tn5 insertion analysis.

Results: This study encapsulated 3,096 multiome RWPE-1 nuclei cultured in serum-free condition, 6,978 and 8,487 multiome LNCaP nuclei treated with methanol (LNCaP-M) or dihydrotestosterone (DHT, LNCaP-D). Our analysis captured 210,990, 264,802, and 278,154 ATAC-seq peaks from RWPE-1, LNCaP-M, and LNCaP-D by macs2 narrow peak model (FDR q<0.01), which included over 90% peaks identified by bulk ATAC-seq datasets. Through downsampling linkage analysis, we identified 1,595, 2,507, and 4,183 linkage associations between ATAC-seq and RNA-seq from the above three experiments, respectively. Scatter plots showed that the correlation coefficient of the linkage association was negatively associated with the distance between ATAC-seq and RNA-seq signals. By comparing to bulk ChIP-seq data from RWPE-1 cells, we confirmed that the single cell ATAC-seq peaks in linkages were co-localized at histone modified regions, including H3K4me1 (25.7%), H3K27ac (14.3%), and H3K4me3 (4.9%). JASPAR database (634 motifs) search found that the Tn5 footprints surrounding AR, NR3C1, and NR3C2 motifs in LNCaP-D showed significantly higher enrichment than that of the LNCaP-M group, indicating increased androgen receptor DNA binding following DHT treatment. Interestingly, in RWPE-1 cells harboring TP53 p.P72R polymorphism (rs1042522), we found differential Tn5 footprints surrounding TP53 binding motif between its ATAC-seq subclusters at whole genome scale.

Conclusion: Our preliminary data in prostate cell lines outlined a regulatory landscape of single-cell resolution. Multiomic single cell sequencing will help locate regulatory elements and target genes.

Citation Format: Yijun Tian, Alex Soupir, Liang Wang. Multiomic single cell sequencing identified regulatory elements in prostate cells [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 782.
Abstract 797: Role of ESR1 mutation in recurrence of tumorigenesis after therapeutic radiation
Authors: Udden S. M. Nashir , Alluri Prasanna G.
Published: June 2022

Acquired mutations in ESR1 gene that encodes for Estrogen Receptor (ER) are a common mechanism of acquired resistance to endocrine therapies in breast cancer. However, the role of such mutations in modulation of cell’s response to radiation therapy is poorly understood. In this study, we define the role of ESR1 mutations in altering response to radiation therapy in pre-clinical models of ER-positive breast cancer. To investigate whether ESR1 mutations influence cell’s response to radiation therapy, we employed ER-positive MCF7 and T47D cell lines which have been genome edited to knock-in two of the most common ESR1 mutations, such as Y537S and D538G, found in breast cancer patients. Clonogenic survival assays were carried out to characterize differences in radiation sensitivity of these cells. g-H2AX and Comet assays were employed to study the differences in residual unrepaired DNA damage following radiation in these cell lines. MCF7 cell line-derived xenografts harboring the wild type or mutant ESR1 were employed to study differences in radiation resistance of these tumors in vivo. We found that ER-positive cell lines harboring ESR1 mutations exhibited profound resistance to radiation therapy compared to cell lines harboring the wild type ESR1. These radioresistant cell lines also demonstrated significantly less unrepaired DNA damage following radiation therapy. We observed that mice harboring ESR1 mutant xenografts showed remarkable resistance to radiation therapy. We also observed that ESR1 mutations, which confer resistance to endocrine therapies, also confer profound resistance to radiation therapy. Our findings suggest that oligometastatic ER-positive breast cancer patients who are being treated with radiation therapy (such as those enrolled in NRG BR002 trial) may show significant differences in response to radiation therapy based on their ESR1 mutation status. Recent studies are also exploring the use of neo-adjuvant endocrine therapy in non-metastatic ER-positive breast cancer patients. It is conceivable that some of these patients may acquire ESR1 mutations under selective pressure of neoadjuvant endocrine therapy and may respond poorly to subsequent adjuvant radiation therapy. Overall, our findings support potential personalization of radiation treatments in breast cancer patients based on ESR1 mutation status, heralding an era of precision radiation oncology.

Citation Format: S. M. Nashir Udden, Prasanna G. Alluri. Role of ESR1 mutation in recurrence of tumorigenesis after therapeutic radiation [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 797.