bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3282: Regorafenib enhance PD-L1 expression via ATM activation and evaluate the therapeutic efficacy of combining PD-L1 antibody on bladder cancer
Authors: Pai Kai-Jen , Chiang I-Tsang , Hsu Fei-Ting , Tsai Jai-Jen
Published: April 2023

Bladder cancer is one of the most common cancer in urinary system in Taiwan. According to Ministry of Health and Welfare’s research, bladder cancer plays 15th leading causes of death in Taiwan in 2020. Recently immune checkpoint inhibitor (ICI) therapy show several benefits on preventing T cell exhaustion, accumulating evidences showed ICI therapy still remain some limitation, including unable to regulated immunosuppressor cells and tumor microenvironment (TME) related elements which might lead to ICI therapy dysfunction. Recently, study report that DNA damage may activate the innate immune response and improve ICI efficacy. Thus, regulating DNA damage or illustrate the role of DNA damage related factor may bring the twilight to immunotherapy. To identify the possible combination methods with ICIs, we used MB49 cells and animal model to test the combination property of regorafenib (a multiple receptor tyrosine kinase inhibitor) and 10F.9G2 (a PD-L1 monoclonal antibody). Our research illustrated the mechanism between regorafenib and PD-L1, we proved that regorafenib-induced regulated PD-L1 is associated with ATM activation. TUENL assay showed regorafenib are able to activate ATM by causing DNA break and thus enhance the expression of PD-L1 on tumor surface. This effect could be an important step to open the interaction potential of PD-L1 antibodies and thus enhance its efficacy. Through the interaction analysis of miRNA targeting gene (RNA22), we found that IDO1 mRNA could specifically interact with endogenous miR-124. Here, the propose mechanism in our research is that regorafenib may downregulate IDO1 expression via increasing of endogenous miR-124 expression. In animal model, the combination therapy showed extraordinary therapeutic effect, not only dramatically decreased tumor volume, but also upregulating the positive TME components, such as CD8+T cells and M1 macrophage. Though regorafenib may induce PD-L1 expression, regorafenib still showed several therapeutic benefits, including downregulating immunosuppressive factors, such as IDO1, VEGF, and suppressing the accumulation of regulatory T cells, myeloid-derived suppressor cells and also repolarizing macrophage from M2-to-M1⋯etc. In conclusion, this study points out the combination therapy of regorafenib and anti-PD-L1 has present several benefits to bladder cancer patients and is associated with miR-124/ATM signaling.

Citation Format: Kai-Jen Pai, I-Tsang Chiang, Fei-Ting Hsu, Jai-Jen Tsai. Regorafenib enhance PD-L1 expression via ATM activation and evaluate the therapeutic efficacy of combining PD-L1 antibody on bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3282.
Abstract 3286: B-hCD94/hNKG2A mice provide a preclinical tool for evaluating therapeutic antibodies targeting human NKG2A
Authors: Shang Chengzhang , He Fang , Kuraguchi Mari , Zhang Jing , Lin Qingcong
Published: April 2023

The immune cell checkpoint NKG2A and its coreceptor, CD94, are expressed by many circulating NK cells and by a subset of CD8 T cells in tumors. When CD94/NKG2A recognizes the HLA-E-peptide complex, the protein tyrosine phosphatase SHP-1 is recruited to the signaling synapse, resulting in the delivery of inhibitory signals to the effector cells and inhibition of their immune activities. In preclinical mouse models, blocking antibodies against NKG2A restores the reactivity of these effector cells, resulting in better suppression of tumor growth. Thus, NKG2A targeting is currently being evaluated in the clinic to evaluate tumor cell killing. To evaluate the in vivo efficacy of anti-NKG2A in mice, we successfully generated a humanized CD94/NKG2A double knock-in mouse strain (B-hCD94/hNKG2A mice), in which the mouse CD94 and NKG2A genes are replaced by the corresponding human CD94 and NKG2A genes in situ in cis, respectively. Human CD94 and NKG2A mRNA and proteins were exclusively detectable by flow cytometry in homozygous humanized mice, confirming successful generation of the model. This model was used to demonstrate in vivo efficacy of monalizumab analog in suppressing the growth of MC38 tumor cells expressing human HLA-E (B-hHLA-E MC38). Furthermore, dual blockade of monalizumab and PD-L1 further suppressed B-hHLA-E MC38 tumor growth. Together, these data indicate that this double humanized model is an effective tool for evaluating novel anti-human NKG2A therapies, and warrants the generation of a quadruple humanized model (B-hPD-1/hPD-L1/hCD94/hNKG2A mice) to further evaluate the synergy of drugs blocking each checkpoint.

Citation Format: Chengzhang Shang, Fang He, Mari Kuraguchi, Jing Zhang, Qingcong Lin. B-hCD94/hNKG2A mice provide a preclinical tool for evaluating therapeutic antibodies targeting human NKG2A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3286.
Abstract 33: Humanized MOLM-13 AML model as a versatile tool to study immune-activating agents
Authors: Castro Carla , Metzger Philipp , Obodozie Cynthia , Weber Holger
Published: April 2023

Acute myeloid leukemia (AML) is the most common acute leukemia and second most common form of blood cancer in children and adolescents. Despite progress in understanding the biology of AML, therapeutic intervention plans have not changed nor outcomes significantly improved over the last years. Current AML treatment consists of intensive chemotherapy followed by stem cell transplantation. While initially many patients respond well to chemotherapy, relapse rates are high and still associated with poor prognosis. Evidence suggests that chemo-resistant leukemic stem cells (LSCs) have the ability to reinitiate and sustain the disease. Therefore, novel therapies are urgently needed to improve AML treatment and well-characterized in vivo models modelling this disease are a prerequisite for improving the current state.

At Reaction Biology, we have used NOG and NOD-SCID mice to establish a humanized MOLM-13 AML model concomitantly engrafted with human T cells. Luciferase positive MOLM-13_Luc AML cells were injected both in naïve mice and mice pre-treated with Cyclophosphamide. Thereafter, tumor growth was monitored by bioluminescence measurements to track disseminated tumor burden and isolated T cells were applied upon tumor growth detection (generally observed on day 8 or 15 post-tumor cell inoculation for NOG or NOD-SCID mice, respectively). For each of the inoculations, fresh T cells were obtained by negative selection from buffy coat derived PBMCs with a purity of >95 %. Following administration of human T cells, animals were monitored daily, including the collection of animal weights (3x/w), and tumor burden (2x/w) via in vivo bioluminescence imaging technique.

MOLM-13_Luc tumor cells grew rapidly in all NOG mice with termination of the last animals as early as day 17. Neither Cyclophosphamide pre-treatment, nor the addition of T cells influenced tumor growth or prolonged survival. In Cyclophosphamide pre-treated NOD-SCID mice, onset of tumor growth was observed to initiate homogenously at day 13 post inoculation (with applied T cells being ineffective in reducing tumor burden), whereas tumor growth in non-pre-treated animals was heterogenous and delayed by more than 7d. The presence of T cells affected tumor growth even further. Beside T cell addition, also the application of NK cell therapies were successfully investigated (data not shown).

Taken together, the MOLM-13_Luc AML tumor model is found to be suitable for the investigation of bi-specific engagers using isolated T or NK cells.

Citation Format: Carla Castro, Philipp Metzger, Cynthia Obodozie, Holger Weber. Humanized MOLM-13 AML model as a versatile tool to study immune-activating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 33.
Abstract 34: A clinically-relevant orthotopic endometrial carcinosarcoma mouse model using human patient-derived organoids
Authors: Katcher Arielle , Chung Charlie , Gorman Megan , Yueh Brian , Lyons Scott , Merrill Joseph , Garcia Libia , Frimer Marina , Goldberg Gary L. , Beyaz Semir
Published: April 2023

Uterine carcinosarcoma (CS) is a rare but aggressive endometrial malignancy. Like the other subtypes of endometrial cancer, the incidence of CS has been increasing and it is projected to continue to rise. Additionally, there is racial disparity seen in African American women diagnosed with CS. African American women have an increased incidence of CS compared to other racial/ethnic groups. They also have an overall increased mortality related to endometrial cancer. Compared to other histologic subtypes of endometrial cancer, CS remains rare and understudied on a molecular and cellular basis. It remains under-represented in clinical trials. Therefore, the current therapies available for CS are extrapolated from those treatments used in the other subtypes of endometrial cancer. There is a paucity of data surrounding targeted therapies for CS resulting in worse outcomes for patients who are diagnosed with this aggressive and lethal cancer. We have developed a clinically relevant, orthotopic CS murine injection model by injecting human CS patient-derived organoids (PDO) from diverse ancestries into the endometrium of immunocompromised mice. Tumor progression in the mice was monitored with physical examination, PET/CT scan, contrast enhanced CT scan, and ultrasound. The tumor was established after three months and was visualized using various imaging techniques. The mice were sacrificed and the diagnosis of invasive CS was confirmed by macroscopic, histologic and genomic analyses. Our orthotopic CS PDO transplantation model has many potential applications for studying tumor biology and the response to treatment for CS in the pre-clinical setting. This may help identify targeted therapy for CS and potentially advance to Phase 1 human clinical trials.

Citation Format: Arielle Katcher, Charlie Chung, Megan Gorman, Brian Yueh, Scott Lyons, Joseph Merrill, Libia Garcia, Marina Frimer, Gary L. Goldberg, Semir Beyaz. A clinically-relevant orthotopic endometrial carcinosarcoma mouse model using human patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 34.
Abstract 3464: Dynamics of resident memory (Trm) cell responses between skin and draining lymph nodes during primary and recall responses to melanoma
Authors: Messier Cameron , Khatwani Nikhil , Hawkes Aaron , Huang Yuyang (Poppy) , Turk Mary Jo
Published: April 2023

Tissue resident memory (TRM) cells reside durably in tissues where they serve as a vital component of protective anti-tumor immunity. In mice, TRM cells are defined by their expression of tissue retention markers such as CD69 and CD103, and lack of egress markers such as CD62L, CCR7 and S1PR1. We previously showed that depletion of regulatory T cells induces the generation of protective CD8 Trm cells in skin and draining LNs of B16 melanoma tumor-excised mice. While tumor specific Trm cells were shown to stably reside in tissues at steady state, little is known about their migration between tissue compartments and circulation during primary and recall responses to melanoma. Using gp100-specific transgenic pmel CD8 T cells expressing a Kaede-GFP photoconvertible marker, we investigated the trafficking patterns of tumor Ag specific CD8+ T-cells that were labeled in situ by exposure to UV light in the skin. During primary tumor growth, we observed pronounced movement of antigen-experienced pmel cells through skin to tumor-draining LNs. This trafficking behavior could be inhibited by treatment with FTY720, indicating its S1PR-dependence. Such movement was no longer evident at memory timepoints. However, re-challenge with B16 cells in the dermis induced rapid pmel T cell egress to both LNs and circulation. Recallenge-mediated Trm cell skin egress was confirmed using anti-Thy1.1 monoclonal antibody to deplete circulating and lymphoid memory, while preserving skin TRM. Here we found that skin TRM proliferate and transiently increase expression of Tbet, but go on to generate a durable secondary Trm response in lymph nodes. These data highlight T cell movement between tissues during Trm programming and recall responses to cancer, and shed light on a potential mechanism for the generation TRM populations in tumor-draining lymph nodes. Moreover, our results highlight the potential for retrograde movement of Trm responses from peripheral tissue reservoirs to participate more broadly in systemic anti-tumor immunity.

Citation Format: Cameron Messier, Nikhil Khatwani, Aaron Hawkes, Yuyang (Poppy) Huang, Mary Jo Turk. Dynamics of resident memory (Trm) cell responses between skin and draining lymph nodes during primary and recall responses to melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3464.
Abstract 3479: Modeling epigenetic lesions that cause gliomas
Authors: Rahme Gilbert J. , Javed Nauman M. , Puorro Kaitlyn L. , Xin Shouhui , Hovestadt Volker , Johnstone Sarah E. , Bernstein Bradley E.
Published: April 2023

Epigenetic lesions that disrupt gene regulatory elements and expression are increasingly recognized as potential drivers of human cancers. However, we currently lack the in vitro and in vivo models required to functionally validate such lesions and their tumorigenic impact. Here we model aberrations that arise in Isocitrate Dehydrogenase (IDH) mutant lower-grade gliomas, which exhibit profound DNA hypermethylation as a direct consequence of mutant IDH. DNA hypermethylation may promote gliomagenesis by silencing tumor suppressor genes or, alternatively, by activating proto-oncogenes through disruption of CCCTC-binding factor (CTCF) insulators. CTCF insulator sites define the three-dimensional shape of the genome by dictating the boundaries of topologically associated domains (TADs). Enhancers and promoters can interact when located in the same TAD but are restricted from interacting across different TADs. In IDH mutant gliomas, CpG sites around CTCF binding sites are frequently methylated, effectively compromising CTCF binding and thus TAD organization, allowing for cross-TAD interactions and aberrant activation of genes. We discovered a CTCF insulator downstream of the PDGFRA proto-oncogene that is recurrently disrupted in IDH mutant gliomas. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce Pdgfra, thereby increasing proliferation. In contrast, insulator disruption did not affect Pdgfra expression in neural progenitor cells (NPCs), which lack the enhancer. We also model a second recurrent epigenetic lesion in IDH mutant gliomas, the methylation-dependent silencing of the CDKN2A tumor suppressor. We show that inactivation of Cdkn2a/p19ARF by de novo promoter methylation or mutation drives OPC proliferation and cooperates with Pdgfra insulator loss. Finally, we use lentiviruses to coordinately inactivate the Pdgfra insulator and Cdkn2a in mouse corpus callosum, resulting in low-grade gliomagenesis in vivo with histological features reminiscent of human IDH mutant gliomas. Our study recapitulates recurrent epigenetic lesions in mouse models and demonstrates that the combination of Pdgfra activation and Cdkn2a silencing can transform OPCs in vitro and drive gliomagenesis in vivo.

Citation Format: Gilbert J. Rahme, Nauman M. Javed, Kaitlyn L. Puorro, Shouhui Xin, Volker Hovestadt, Sarah E. Johnstone, Bradley E. Bernstein. Modeling epigenetic lesions that cause gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3479.
Abstract 3485: Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity
Authors: Patil Ajinkya , Strom Amy R. , Collings Clayton K. , Paulo Joao A. , Wauer Tobias , Sankar Akshay , St.Laurent Jessica D. , Cervantes Kasey S. , Gygi Steven P. , Brangwynne Clifford P. , Kadoch Cigall
Published: April 2023

The mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes collectively represent one of the most frequently mutated cellular entities in cancer, second only to TP53. Mutations across the 29 human genes that encode mSWI/SNF complex subunits occur in over 20% of human cancers, with mutations affecting ARID1A and ARID1B, the largest BAF subunits, being the most frequent. However, the functional contributions of these subunits, particularly their commonly mutated N-terminal intrinsically disordered regions (IDRs) and a highly conserved ARID DNA-binding domain, to BAF function remain poorly understood. Here, we demonstrate that the IDRs of ARID1A/B, coupled with the ARID domain, drive biomolecular condensate formation and BAF chromatin localization in cells. We define ARID1A/B IDRs as two-part systems, facilitating homotypic BAF complex interactions (i.e., valence generated by localized condensation) and heterotypic complex interactions, that together establish a highly specific, sequence-encoded protein interaction network within condensates. Both types of interactions are required for appropriate genome-wide targeting of BAF complexes, DNA accessibility generation, and appropriate gene expression. Replacement of the ARID1A N-terminal IDR with IDRs derived from two unrelated proteins FUS and DDX4, rescues generic condensation of BAF but not chromatin occupancy, DNA accessibility, and heterotypic interactions, highlighting the sequence-specificity embedded in the IDR of ARID1A. Taken together, these data establish a role for the largest and most frequently perturbed IDRs within a major chromatin remodeler and explain how biomolecular condensate formation enables both genomic localization and functional partner recruitment. Furthermore, these findings lay the groundwork for mapping IDR sequence specificity or “grammar”, that dictates the co-condensation network formation, and suggests that targeted disruption of these mechanisms may represent new targeted therapeutic opportunities across multiple cancers.

Citation Format: Ajinkya Patil, Amy R. Strom, Clayton K. Collings, Joao A. Paulo, Tobias Wauer, Akshay Sankar, Jessica D. St.Laurent, Kasey S. Cervantes, Steven P. Gygi, Clifford P. Brangwynne, Cigall Kadoch. Intrinsically disordered regions of the ARID1A/B tumor suppressors encode an interaction network within biomolecular condensates that directs mSWI/SNF chromatin remodeler complex activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3485.
Abstract 3500: A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma
Authors: Ali Raja Hussain , Silverman Brianna , Orellana Esteban A. , Kennedy Alyssa , Bowers Caitlin , Gutierrez Ashley , Papke David , Fletcher Christopher D. , Atayde Antonio P. , Shimamura Akiko , Gregory Richard I. , Gutierrez Alejandro
Published: April 2023

Amplification of chromosomal material derived from 12q is pathognomonic in a subset of soft tissue sarcomas (STS), including well-differentiated and dedifferentiated liposarcoma. However, it remains unclear which of the amplified genes are oncogenic drivers, obstacles to rational therapeutic development. We defined a minimal 12q amplicon in human liposarcoma, and tested the oncogenic activity of each recurrently amplified gene to accelerate the onset of AKT-induced STS in a genetically engineered zebrafish model of the disease. This revealed several genes whose overexpression accelerated the onset of AKT-induced STS, including METTL1, best-known for its ability to catalyze methylation of tRNAs. Surprisingly, catalytically defective mutants of METTL1 exhibited oncogenic activity that was at least as potent as the wild-type gene in zebrafish and human STS, indicating enzyme-independent oncogenic functions. We found that METTL1 promotes ribosome assembly and oncogenic translation independent of its enzymatic activity. In human liposarcoma cells and mouse embryonic fibroblasts, METTL1 overexpression was sufficient to stimulate polysome formation, and this effect was phenocopied by catalytically dead mutants of METTL1. METTL1 overexpression also robustly stimulated the rate of protein synthesis in cells (P < 0.0001), and in a cell-free biochemical assay for translation of a reporter mRNA (P < 0.001). The ability of METTL1 to stimulate ribosomal function triggered a therapeutically targetable dependency, because small molecule inhibitors of ribosome biogenesis had potent therapeutic activity against METTL1-amplified (P < 0.0001), but not METTL1 wild-type, sarcoma cells. Thus, METTL1 is a soft tissue sarcoma oncogene that stimulates oncogenic translation independent of its enzymatic activity, an oncogenic function that can be targeted therapeutically.

Citation Format: Raja Hussain Ali, Brianna Silverman, Esteban A. Orellana, Alyssa Kennedy, Caitlin Bowers, Ashley Gutierrez, David Papke, Christopher D. Fletcher, Antonio P. Atayde, Akiko Shimamura, Richard I. Gregory, Alejandro Gutierrez. A noncatalytic role for mettl1 in oncogenic translation in soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3500.
Abstract 3789: Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance
Authors: Ngue Harrison , Shrestha Jitendra , Kim Hyejin , Vasudevan Shobha
Published: April 2023

Up to 90% of failures in chemotherapy treatment are associated with metastatic-related drug resistance. Extracellular vesicles (EVs) are believed to play a role in the spread of chemotherapy resistance through the horizontal transfer of oncoproteins and noncoding RNA species, but the underlying mechanisms for this are not fully understood. Here, we study EV biogenesis, release, and uptake in the context of breast cancer chemoresistance. Specifically, we characterized the bioactive cargoes in EVs secreted by chemoresistant triple negative breast cancer cells and chemoresistant estrogen receptor-positive breast cancer cell lines. Preliminary data show that these EVs contain unusually high levels of circular RNAs, small RNA species formed covalently through backsplicing and are typically noncoding. When chemosensitive cells absorb these circRNA-enriched EVs, they experience a significant increase in resistance phenotypes. Moreover, knockdown of the circRNAs via antisense oligonucleotides results in decreased chemoresistance, suggesting a role of circRNAs in the development of chemotherapy resistance. Previous studies have shown that tumor-derived exosomal circRNAs may confer resistance by sponging microRNAs. We are exploring what the circRNAs do mechanistically to promote tumor survival, and clarifying these mechanisms as a whole will serve to potentially provide therapeutic targets against chemotherapy resistance and cancer recurrence.

Citation Format: Harrison Ngue, Jitendra Shrestha, Hyejin Kim, Shobha Vasudevan. Circular RNA expression is enriched in breast cancer extracellular vesicles and is associated with chemotherapy resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3789.
Abstract 3793: Regulation of miR-124 expression by promoter methylation in CRC and its relationship with KITENIN expression
Authors: Park So-Yeon , Zhou Rui , Gamage Chathurika D. B. , Pulat Sultan , Varlı Mücahit , Kim Hangun
Published: April 2023

The mechanisms that regulate microRNA expression in cancer are not fully understood. However, recently reported data show that the epigenetic mechanism plays an important role in the regulation of microRNA expression. In in this study, we investigated the relationship between miR-124 promoter methylation and target genes in CRC. The expression of miR-124 was increased by Aza treatment and the expression of KITENIN was decreased in CRC cells. First, we examined the methylation status of miR-124 promoter in two normal cell lines and seven CRC cell lines using pyrosequencing assay. As a result, miR-124 promoter was hypermethylated in CRC cells and low in normal cells. Next, we evaluated the DNA methylation status for a miR-124 promoter in paired colorectal normal tissues and colorectal cancer tissues specimens from 10 patients. Overall, methylation of the miR-124 promoter was high in cancer among clinical tissues, and the expression of miR-124 was low in cancer tissues. The expression of the KITENIN was also higher in cancer tissues than in normal tissues. These results prove that DNA hypermethylation contributes to the transcriptional down-regulation of miR-124 in colon cancer, and that the epigenetic silencing of miR-124 in cancer cells modulate the activity of KITENIN. Our findings provide basic data that can be useful for clinical treatment of CRC patients through the mechanism of miR-124 regulation identified as a regulator of KITENIN.

Citation Format: So-Yeon Park, Rui Zhou, Chathurika D. B. Gamage, Sultan Pulat, Mücahit Varlı, Hangun Kim. Regulation of miR-124 expression by promoter methylation in CRC and its relationship with KITENIN expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3793.
Abstract 3885: A secondary Gαq mutation confers resistance to Gαq inhibitors in uveal melanoma
Authors: Ma Jiafang , Wang Meng , Manglik Aashish , Bastian Boris C. , Chen Xu
Published: April 2023

Uveal melanoma (UM) is a rare subtype of melanoma that originates from melanocytes of the choroidal plexus, ciliary body and iris. Despite successful treatment of the primary by radiation or surgery, 50% of UM patients develop metastases, mostly in the liver, as an invariably lethal complication. Over 90% of UM harbor activating mutations in the closely related GNAQ or GNA11 genes, mainly at codons Q209, compromising the GTPase activity. Mutant GNAQ and GNA11 can be successfully targeted by the cyclic depsipeptide YM-254890. To anticipate mechanisms of resistance that might arise under treatment with this novel class of Gαq inhibitors, we generated UM cell lines resistant to YM-254890 using an in-vitro cell culture system. Upon chronic growth suppression of a GNA11Q209L mutant UM cell line, we established 12 YM-254890 resistance clones. We identified a secondary GNA11F75Y mutation in one clone and GNA11Y192H mutations in the other 11 clones. The secondary mutations were present in cis with the original GNA11 Q209L mutations. To validate the functional role of these secondary mutations in conveying resistance, we engineered two double mutants, GNA11Q209L/F75Y and GNA11Q209L/Y192H, and introduced them into 293FT cells respectively and confirmed that both F75Y and Y192H convey resistance to YM-254890 treatment. The proliferation of YM-254890-resistant cell lines was still dependent on GNA11 and downstream PKC/MAPK signaling. Combined inhibition of PKC and MEK synergistically reduced cell viability in YM-254890 resistant UM cells. Analysis of the crystal structure of YM-2548890 in complex with GNAQ predicts both F75Y and Y192H mutations directly affect the binding of YM-254890 to Gαq. Our data suggest that direct targeting mutant GNAQ/11 is promising but will select for secondary mutations within GNAQ/11 that will result in resistance. Combinatorial targeting of other components in the Gαq signaling pathway will increase clinical efficacy.

Citation Format: Jiafang Ma, Meng Wang, Aashish Manglik, Boris C. Bastian, Xu Chen. A secondary Gαq mutation confers resistance to Gαq inhibitors in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3885.
Abstract 396: Role of PHF8 in resistance to melanoma targeted therapies
Authors: Gómez-Muñoz María de los Ángeles , Eskow Nicole , Delclaux Ines , Moubarak Rana , Hernando-Monge Eva
Published: April 2023

Melanoma is a highly prevalent cancer with increasing incidence worldwide. While immune checkpoint inhibitors have drastically improved outcomes for about half of metastatic melanoma patients, many are still resistant to these treatments or develop life-threatening toxicities. Around 50% of melanomas carry BRAF mutations that result in constitutive activation of the MAPK pathway. Therapeutic combinations of BRAF and MEK inhibitors (MAPKi) have proven very efficient against these tumors, but responses are not durable and treatment-resistant recurrence almost inevitably occurs. Our group recently uncovered a role for the histone demethylase PHF8 in melanoma metastasis, and demonstrated that PHF8 directly controls the expression of key components of the TGFβ pathway. Given that TGFβ signaling crosstalks with the MAPK pathway and contributes to resistance to therapy in various tumors, we decided to investigate a potential role for PHF8 in response and resistance to MAPKi therapy in melanoma. To test our hypothesis, we assessed the effect of modulating PHF8 expression in BRAFV600E-mutant cells and their matched MAPKi-resistant derivatives. PHF8 silencing did not affect cell survival or proliferation of parental, MAPKi-sensitive melanoma cell lines, but it re-sensitized resistant cells to MAPKi treatment. Conversely, PHF8 overexpression in melanoma cell lines did not affect their proliferation rate but increased the emergence of resistant clones in BRAFV600E-mutant melanoma cells treated with BRAFi (Dabrafenib) and MEKi (Trametinib) combinations. Next, we assessed the dynamics of PHF8 expression and TGFβ signaling in melanoma cells exposed to MAPKi. Treatment of MAPKi-sensitive cells leads to a fast and complete downregulation of PHF8 protein expression without a change in mRNA levels, paralleled by reduced TGFβ signaling, as evidenced by decreased SMAD2 phosphorylation. However, PHF8 protein expression is restored in MAPKi resistant clones. Therefore, based on our preliminary data, we postulate that PHF8 suppression by the MAPK pathway plays a critical role in melanoma response to targeted therapy and that its restoration contributes to acquisition of resistance. We are currently investigating MAPK-dependent mechanisms controlling PHF8 stability, and testing novel PHF8 inhibitors for their ability to bypass or overcome melanoma resistance to MAPKi. Our studies might provide basis for a novel epigenetic therapy as a strategy to maximize the potential of MAPKi therapy in melanoma treatment.

Citation Format: María de los Ángeles Gómez-Muñoz, Nicole Eskow, Ines Delclaux, Rana Moubarak, Eva Hernando-Monge. Role of PHF8 in resistance to melanoma targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 396.
Abstract 3968: Conophylline-target ARL6ip1 regulates Ran-mediated cellular migration and invasion in human colorectal cancer cells
Authors: Lin Yinzhi , Karnan Sivasundaram , Ito Hideaki , Umezawa Kazuo
Published: April 2023

Introduction: Conophylline is an alkaloid isolated from the leaves of Ervatamia microphylla. We isolated conophylline as an inhibitor of K-Ras functions. It is known to ameliorate various disease models in animals, including cancer, diabetes mellitus, NASH, and hepatic cirrhosis. On the other hand, its molecular target was determined to be ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) using a conophylline-biotin conjugate. ARL6ip1 is located in the endoplasmic reticulum (ER) membrane, and the conophylline-binding domain was determined by deletion mutation analysis. Known functions of ARL6ip1 include inhibition of apoptosis, inhibition of glutamate transporter, and modulation of the ER structure. However, whether ARL6ip1 is involved in the mechanisms of various biological activities of conophylline has not been proven, since knockdown of ARL6ip1 has often failed to change the cellular phenotypes. Therefore, in the present research, we knocked out ARL6ip1 in human colon carcinoma cells by CRISPR-Cas9 and studied the involvement and mechanism of ARL6ip1 in the anticancer activity of conophylline. Ran activity was measured by a pull-down assay with Ran-GTP antibody.

Materials and Methods: Conophylline was isolated from the leaves of Ervatamia microphylla. We employed HCT116 and DLD1 cells as human colorectal cancer cells. Tumorigenicity was measured by soft agar colony formation. Cellular migration was measured by a wound healing assay and cell tracking analysis. Cellular invasion was measured by a Matrigel chamber assay.

Results: Conophylline inhibited soft agar colony formation in HCT116 and DLD1 cells. It inhibited migration and invasion in HCT116 and DLD1 cells at nontoxic concentrations. Knockout of ARL6ip1 also decreased tumorigenicity, migration, and invasion in HCT116 and DLD1 cells. The mechanistic study was carried out with HCT116 cells. ARL6 and Ran are both G-proteins and are reported to interact with ARL6ip1. Although knockdown of ARL6 did not change the cell migratory activity, knockdown of Ran inhibited the migration. Conophylline inhibited the interaction of ARL6ip1 to Ran in the proximity ligation assay. Moreover, conophylline inhibited the Ran activity.

Conclusion: CRISPR-Cas9 knockout of ARL6ip1 showed a similar anticancer activity as treatment with conophylline. It is likely that the anticancer activity of conophylline would be mediated by the ARL6ip1-Ran system. Thus, ARL6ip1 would be a useful molecular target for the treatment of cancer.

Citation Format: Yinzhi Lin, Sivasundaram Karnan, Hideaki Ito, Kazuo Umezawa. Conophylline-target ARL6ip1 regulates Ran-mediated cellular migration and invasion in human colorectal cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3968.
Abstract 3995: Trastuzumab deruxtecan, antibody-drug conjugate targeting HER2, effectively inhibits growth of patient-derived xenograft model with CIC-rearranged sarcoma
Authors: Kojima Yuki , Yagishita Shigehiro , Sudo Kazuki , Shimoi Tatsunori , Iwata Shintaro , Watanabe Shun-ichi , Ogawa Chitose , Yoshida Akihiko , Yatabe Yasushi , Yonemori Kan , Hamada Akinobu
Published: April 2023

Background: Capicua-double homeobox 4 (CIC-DUX4)-rearranged sarcomas (CDS) are extremely rare and highly aggressive sarcomas. There is no standard therapy for the patients with advanced CDS, and therapeutic development is needed. We evaluated preclinical efficacy of trastuzumab deruxtecan (T-DXd), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient-derived xenograft (PDX) models with CDS.

Methods: Patient-derived tumor tissue was transplanted into subcutaneous around the flank of female NOG mice, which were treated with vehicle or T-DXd (3 mg/kg, intravenous, Day0) when the mean tumor volume reached 200 mm3. Tumor volume was assessed twice weekly for 3 weeks to assess the efficacy of T-DXd.

Results: This study included 3 CDS-derived PDXs. HER2 expression was low in one PDX and not expressed in two PDXs. One PDX was established from a specimen at initial diagnosis, two were established from specimens after prior-chemotherapy. T-DXd demonstrated significant tumor growth delay compared to vehicle in all PDX models investigated. One PDX with no efficacy was HER2-negative and had a treatment history of topoisomerase I inhibitor. In contrast, PDX of HER2-negative topoisomerase I inhibitor-resistant Ewing sarcoma was also administered T-DXd under the same conditions and was found to be refractory.

Conclusion: The present study showed that a therapeutic potential of T-DXd in CDS patients, including HER2-negative.

Citation Format: Yuki Kojima, Shigehiro Yagishita, Kazuki Sudo, Tatsunori Shimoi, Shintaro Iwata, Shun-ichi Watanabe, Chitose Ogawa, Akihiko Yoshida, Yasushi Yatabe, Kan Yonemori, Akinobu Hamada. Trastuzumab deruxtecan, antibody-drug conjugate targeting HER2, effectively inhibits growth of patient-derived xenograft model with CIC-rearranged sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3995.
Abstract 400: TIP60-mediated regulation of DNp63a is associated with cisplatin resistance
Authors: Hira Akshay , Stacy Andrew J. , Zhang Jin , Kemp Mike , Kadakia Madhavi P.
Published: April 2023

About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. The chemotherapeutic drug, Cisplatin is often used to treat squamous cell carcinoma (SCC) patients, but low response rates and disease recurrence is common. ΔNp63α, a member of the p53 family of transcription factors, is overexpressed and considered oncogenic in non-melanoma skin cancer where it regulates cell survival, promotes proliferation and inhibits cell apoptosis. ΔNp63α has also been shown to promote resistance to cisplatin by transcriptionally regulating several DNA damage response (DDR) genes. A previous study from our lab showed that the histone acetyltransferase (HAT) TIP60 promotes SCC proliferation by positively regulating ΔNp63α protein levels in manner dependent on the catalytic activity of TIP60. This finding suggested that TIP60 may contribute to the failure of platinum-based drugs in SCC and led us to hypothesize that TIP60-mediated acetylation of ΔNp63α regulates its stability and transcriptional activity to promote chemoresistance. Silencing endogenous TIP60 led to a decrease in ΔNp63α transcript and protein levels in multiple SCC cell lines, indicating the positive regulation of ΔNp63α by TIP60 is not cell-line specific. Further, TIP60 levels positively correlated with ΔNp63α stability, protein levels and cisplatin resistance. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α and TIP60 induced G2/M arrest, increased cell-death and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Finally, we demonstrated that ΔNp63α and TIP60 levels positively correlated with DNA repair capacity and negatively correlated with cisplatin-DNA adduct formation. Silencing of either TIP60 or ΔNp63α enhanced cisplatin-DNA adduct formation and significantly reduced expression of genes involved in DDR. Taken together, our data indicate that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance through regulation of genes involved in DNA damage repair. Our findings suggest that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC other epithelial cancers.

Citation Format: Akshay Hira, Andrew J. Stacy, Jin Zhang, Mike Kemp, Madhavi P. Kadakia. TIP60-mediated regulation of DNp63a is associated with cisplatin resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 400.
Abstract 4016: Discovery of a small-molecule EGFR inhibitor that can potently target various EGFR mutants, including C797S mutant, in vitro and in vivo
Authors: Jeon Yeejin , Lee Kiram , Jang Anna , Kim Miyeon , Yoon Kyeong Jin , Lee Yeri , Kim Dongsu , Kim Donggeon , Park Dohyun , Lim Sang Kyun , Choi Sung Pil
Published: April 2023

EGFR is one of receptor tyrosine kinases (RTKs) and, upon ligand binding, turns on the downstream signals that include oncogenic RAS/MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT pathways. EGFR gene mutations, such as Exon 19 deletion (Del19) and L858R mutations, induce abnormal activation of the EGFR protein even in the absence of the ligand leading to various cancers. EGFR mutations are found in non-small cell lung cancer (NSCLC) patients with 10-15% frequency (approximately 50% in Asian patients).

Starting from Del19 and L8585R mutations, additional mutations in the EGFR gene occur during the treatment with 1st/2nd/3rd generation EGFR inhibitors, resulting in Del19/T790M, L858R/T790M, Del19/T790M/C797S, L858R/T790M/C797S, Del19/C797S, and L858R/C797S mutations. While double mutations that include the T790M mutation have been efficaciously treated with Osimertinib, there are currently no approved therapies that can effectively target the C797S-containing mutants.

Here we present preclinical data showing a small-molecule inhibitor that effectively disables EGFR mutants, including Del19/T790M/C797S, L858R/T790M/C797S, Del19/C797S, and L858R/C797S mutants. High potency and selectivity were confirmed by in vitro kinase assays and cellular assays. Outstanding anti-cancer activity was observed in mouse xenograft models with various EGFR mutations and, importantly, it was inactive in the EGFR wild-type model. With confirmed brain penetrance through in vivo studies, our potent EGFR inhibitor may provide a great therapeutic potential for patients with EGFR mutation-driven NSCLC.

Citation Format: Yeejin Jeon, Kiram Lee, Anna Jang, Miyeon Kim, Kyeong Jin Yoon, Yeri Lee, Dongsu Kim, Donggeon Kim, Dohyun Park, Sang Kyun Lim, Sung Pil Choi. Discovery of a small-molecule EGFR inhibitor that can potently target various EGFR mutants, including C797S mutant, in vitro and in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4016.
Abstract 4017: TAS2940 inhibits intracranial tumor growth and prolongs survival in HER2-aberrant and EGFR-amplified patient-derived xenograft models
Authors: Han Jing , Mahendra Mikhila , Gandhi Poojabahen , Daniele Joseph R. , Carrillo Caroline C. , Bivona Benjamin J. , Feng Ningping , Heymach John V. , Meric-Bernstam Funda , Oguchi Kei , Mizuarai Shinji , Heffernan Timothy P. , Vellano Christopher P. , Marszalek Joseph R.
Published: April 2023

Patients with brain tumors and metastases have poor prognosis and overall survival rates despite the advancements in neurosurgery, radiotherapy, and chemotherapy. Genomic alterations in HER2 are present in brain metastases of breast cancer (BC; ~20%) and non-small cell lung cancer (NSCLC; 13-20%), and EGFR alterations occur frequently in glioblastoma multiforme (GBM; >50%). Despite the advancements in standard of care options, optimal treatment management for these patients remains an unmet medical need. Recent evidence suggests activity of systemic therapy for immune and targeted therapies in the brain, including agents targeting HER2/EGFR. HER2-targeted tyrosine kinase inhibitors lapatinib, neratinib, and tucatinib and the HER2-targeted antibodies trastuzumab and pertuzumab, in combination with chemotherapy, have been shown to improve survival of patients with HER2 overexpressing BC in the presence of brain metastases. The limited penetration of these compounds into the CNS, however, limits their efficacy. TAS2940 is an irreversible pan-ErbB inhibitor with greater brain-penetrability than poziotinib, tucatinib, and neratinib. Here, we demonstrate that TAS2940 induces downregulation of phosphorylated HER2/EGFR, reduces tumor burden, and promotes a significant increase in survival in intracranial xenograft mouse models with HER2-amplification (BC), HER2-Exon20 insertion mutation (NSCLC), and EGFR-amplification (GBM). These promising preclinical data highlight potential novel therapeutic strategies for patients with EGFR-aberrant GBM and brain metastases harboring HER2/EGFR alterations, and may help support the advancement of the ongoing first-in-human clinical trial (NCT04982926) for TAS2940 in solid tumors with EGFR and/or HER2 alterations.

Citation Format: Jing Han, Mikhila Mahendra, Poojabahen Gandhi, Joseph R. Daniele, Caroline C. Carrillo, Benjamin J. Bivona, Ningping Feng, John V. Heymach, Funda Meric-Bernstam, Kei Oguchi, Shinji Mizuarai, Timothy P. Heffernan, Christopher P. Vellano, Joseph R. Marszalek. TAS2940 inhibits intracranial tumor growth and prolongs survival in HER2-aberrant and EGFR-amplified patient-derived xenograft models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4017.