Aberrant Activation Research Articles

Abstract 2065: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity

Abstract Adenosine Deaminase Acting on RNA (ADAR) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif. The ADAR1 gene encodes both a universally expressed isoform (p110) and an interferon-inducible isoform (p150), which plays a role in innate immunity by mediating interferon signaling. In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating downstream dsRNA sensors RIG-I and MDA5, which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in several types of cancer and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies such PD-1/PD-L1 blockade and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development. We have developed and validated a high-throughput, cell-based assay for measuring ADAR1 editing activity. Several ADAR editing reporter constructs were designed and evaluated for their relative response to ADAR1 over-expression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a firefly luciferase gene. We show that increasing transient expression of ADAR1 led to proportional increased activity of the downstream firefly luciferase, indicating dose-dependent transcript editing by ADAR1. Selected reporters were used in conjunction with ADAR2 overexpression to identify an ADAR1-biased reporter. Thus, a reporter with low luciferase activity in response to ADAR2 expression was chosen to establish stable HEK293 cell lines (which express low levels of ADAR1) with and without constitutively expressed ADAR1 protein. ADAR1-overexpressing HEK293 cells displayed high luciferase activity, in accordance with high ADAR1-mediated editing of the reporter transcript, which was decreased in response to siRNA-mediated knockdown of ADAR1 expression or pharmacological inhibition of ADAR1 activity. Sequencing of the reporter RNA confirmed that changes in reporter activity were correlated with editing of the RNA transcript. Finally, these validated ADAR1 editing reporter cell lines were further engineered to constitutively express Renilla luciferase under the control of a CMV promoter, serving as an internal control for the toxicity of ADAR1 inhibitors. In conclusion, our robust cell-based assay provides a well-validated resource to identify modulators of ADAR1 editing activity in a cellular context. Citation Format: Valerie Sapp, Jim Bilakovics, Ava Burr, Fernando Martins, Pavel Shashkin, Veronique Baron, Henry Zhu, Junguk Park. Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2065.

Abstract 607: Development of a novel, selective, and potent inhibitor of RON kinase

Abstract Purpose: Receptor d’origine nantais (RON) is a highly conserved transmembrane protein expressed at low levels in healthy adult tissues of epithelial origin and at various levels in immune cells such as macrophages. Aberrant activation of RON has been described in many solid tumors, and it contributes to tumorigenesis by modulating the immune tumor microenvironment, activating numerous oncogenic signaling pathways, and protecting tumor cells under stress. The overexpression of RON protein and the subsequent generation of oncogenic variants are mainly responsible for the persistent activation of downstream signaling pathways. In breast cancer, more than 80% of primary cancer samples are positive for RON expression, with overexpression reported in approximately 50% of cases. Importantly, tumoral RON expression correlates with increased breast cancer progression, metastasis, and poor prognosis independent of molecular subtype. We hypothesize that a small molecule inhibitor of RON that can target both full-length and truncated isoforms will promote anti-tumor immunity within the tumor microenvironment and block pro-metastatic signaling pathways. Herein, we report the development of a novel, orally bioavailable, selective, and potent inhibitor of RON kinase. Methods: With structure-guided and iterative medicinal chemistry approaches, we identified a novel chemical entity that potently inhibited RON kinase activity. Further optimization resulted in the identification of ZB-60 with improved potency, selectivity, and desirable drug-like properties. In vitro kinase assays were performed using ADP-Glo RON kinase assay kit (Promega). Changes in phosphorylation of RON was demonstrated by Western blotting. Plasma pharmacokinetics (PK) was conducted in mice to measure exposures and to determine the oral bioavailability of ZB-60. In vivo efficacy of ZB-60 was evaluated in syngeneic mouse models of breast (EMT6) and colorectal (CT26) cancer. Results: ZB-60 inhibited RON kinase activity with an IC50 of 56 nM and showed greater than 40-fold selectivity against MET and other closest members of the tyrosine kinase family. In ZB-60 treated breast cancer cells, there was a dose dependent reduction of RON phosphorylation and downstream signaling. PK analysis showed a high oral bioavailability with low clearance. In vivo evaluation of ZB-60 showed inhibition of tumor growth in EMT6 and CT26 models. Ongoing studies are focused on testing ZB-60 in combination with checkpoint inhibitors. The results from these studies along with ADME-TOX and pharmacokinetics will be presented. Conclusion: ZB-60 has target specific activity and exhibits favorable drug-like properties. Citation Format: Mohan Rao Kaadige, Trason Thode, Jaime Fornetti, Alexis Weston, Serina Ng, Tithi Ghosh-Halder, Taylor Bargenquast, Brian Durbin, Srinivas Kasibhatla, Raffaella Soldi, Alana Welm, Sunil Sharma. Development of a novel, selective, and potent inhibitor of RON kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 607.

Abstract 5728: YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors

Abstract Metastatic breast cancer (BC) is the 2nd leading cause of death in women in the US, annually accounting for more than 43,000 deaths and 281,000 new cases of invasive BC. Amongst genetically distinct BC subtypes, those classified as being “triple-negative” (TNBC) are especially devastating due to their highly metastatic behavior, their propensity to recur rapidly, and their low response to standard-of-care therapies. In fact, the acquisition of chemoresistant phenotypes represents the main cause of disease recurrence, metastasis, and death in TNBC patients. Currently, the molecular mechanisms that regulate TNBC progression and metastasis remain unknown, as does the way these metastatic tumors acquire resistance to standard-of-care therapies. We recently established YB1 as a novel driver of these deadly TNBC activities, doing so by stimulating the cancer stem cell phenotype, and by disrupting normal cell cycle progression, therefore promoting therapy resistance and metastasis of TNBC tumors. YB1 is a multifunctional protein that acts as a transcription factor of cancer stem cell genes Nanog, Oct and Sox. Moreover, aberrant activation of YB1 contributes to the metastatic progression of several cancers, including TNBC. Our investigations revealed a major role of YB1 in the regulation of several hallmarks of cancer that drive TNBC tumors progression and metastasis, both in vitro and in preclinical mouse models of TNBC tumors. In extending these discoveries, we now show that aberrant YB1 expression activates oncogenic signaling leading to the dysregulation of cell cycle progression through the regulation of the Cyclin D/CDK4/6 complex signaling and the RB pathway. We also report the identification of a novel small molecule inhibitor, SU056, that specifically targets YB1 and inhibits its oncogenic activity in TNBC cell lines. We further show that the SU056-mediated inhibition of YB1, either as monotherapy or in combination with the CDK4/6 inhibitors, has a significant impact on inhibiting TNBC tumor progression and metastasis. In fact, the SU056-CDK4/6 inhibitors combination showed a synergistic effect when compared to monotherapy, at the same time reducing the toxicity associated with CDK4/6 inhibitors. Mechanistically, our studies revealed that genetic or pharmacologic targeting of YB1 inhibits TNBC tumor progression and metastasis through the regulation cyclin D-CDK4/6-RB pathway and blockade of cell cycle progression. Together, our data support the notion that targeting YB1 represents a potential therapeutic option for the treatment of TNBC, which could be enhanced when combined with standard of care treatment modalities. Citation Format: Wei Wang, Lamyae El Khalki, Neelum Yousaf Zai, Justin Szpendyk, Akram Alkrekshi, Bin Su, Khalid Sossey-Alaoui. YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5728.

Abstract 4369: CYP3A5 promotes aggressive and therapeutically resistant prostate cancer by modulating AR and Wnt signaling: Implications for racially disparate clinical outcomes

Abstract Prostate cancer (PC) is the second leading cause of cancer-related death in American men, which disproportionately affects Black/African American (AA) men. The androgen receptor (AR) signaling plays a pivotal role in PC development and is a primary target for intervention in patients with advanced disease. Aberrant activation of AR is also suggested to play a significant role in castration-resistant prostate cancer (CRPC). Despite advancements in anti-androgen therapies with new targeting agents, such as enzalutamide, metastatic CRPC (mCRPC) remains incurable. CYP3A5, a monooxygenase expressed in the prostate, liver and intestine, is involved in drug metabolism and steroid biosynthesis. We earlier reported that intratumoral CYP3A5 activated AR signaling by facilitating its nuclear translocation of AR. More notably, we found a higher CYP3A5 expression in AAs, attributed to the *1 CYP3A5 variant, as compared to non-Hispanic White Americans (NHWA) possessing the *3 variant. Analysis of RNA-seq data using patient tumor samples revealed that elevated CYP3A5 in AA PC patients was associated with Wnt-β catenin signaling activation via TCF4 overexpression. Here, we investigated the role of CYP3A5 in enzalutamide resistance and Wnt signaling by generating enzalutamide-resistant PC cell lines of NHWA (low CYP3A5-LNCaP) and AA-origin (high CYP3A5-MDAPCa2b). RT-qPCR assay was used to examine the changes in AR and CYP3A5 expression between the parental and the enzalutamide-resistant cell lines. The data show heightened CYP3A5 expression in enzalutamide-resistant cells of AA origin (MDAPCa2b/EnzR) but not of NHWA origin (LNCaP/EnzR). Interestingly, MDAPCa2b/EnzR exhibited no change in AR levels whereas it was increased in LNCaP/EnzR cells relative to parental lines. A loss of function assay was performed for siRNA-mediated silencing of CYP3A5 to study its effect on Wnt signaling. RNA sequencing results show that CYP3A5 inhibition in MDAPCa2b downregulated Wnt pathway genes (Wnt5A, Wnt10B, Wnt11, Fzd2, and Dvl3). These observations align with our previous RNA seq results using patient samples where we observed upregulated Wnt-β catenin signaling in high CYP3A5 expressing patient tumor samples. Western analysis post-CYP3A5 inhibition in LNCaP and MDAPCa2b cells revealed upregulated Axin and downregulated LRP6 and Dvl-3, signifying CYP3A5's regulation of the canonical Wnt-β catenin pathway. Additionally, MDAPCa2b saw downregulated Wnt 5A and Wnt 10B, known to influence the Wnt non-canonical pathway. Altogether, our data suggest that *1 CYP3A5 variant in AAs likely contributes to aggressive behavior and therapeutic resistance of PC and serves as a molecular determinant of disparate clinical outcomes. Citation Format: Jake McLean, Ajay P. Singh, Edwin Oh, Ranjana Mitra. CYP3A5 promotes aggressive and therapeutically resistant prostate cancer by modulating AR and Wnt signaling: Implications for racially disparate clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4369.

Abstract 633: Integrin αVβ3 activation by thyroid hormones triggers JAK/STAT oncogenic pathways that promote T cell lymphoma dissemination

Abstract T cell lymphomas (TCL) are a heterogeneous group of lymphoproliferative disorders with poor prognosis. Aberrant activation of JAK/STAT pathway is associated with lymphoma dissemination in TCL patients. Although the FDA approved the use of inhibitors to target this pathway (Ruxolitinib), significant toxic effects have been reported and their use is limited. Thus making an excellent opportunity to study biological factors that regulate the activation of these pathways. In this sense, we recently showed that thyroid hormones (THs) acting through integrin αVβ3 induced TCL proliferation, which is reduced in vitro and in vivo in the presence of integrin αVβ3 pharmacological inhibitor. Our aim is to evaluate the impact of THs on JAK/STAT pathway activation and their implications on anti-lymphoma therapy. For the in vitro assays we used human and murine TCL cell lines. We used EL4 cells and c57bl/6 syngeneic mice for in vivo analysis. Proteomic profiles were evaluated by LC-MS-MS analysis. The GSE58445 database was used for in silico analysis. We found that THs induced STAT1, 3 and 5 phosphorylation in TCL cells and that the integrin αVβ3 inhibitor, Cilengitide (Cile) blunted these effects (p<0.05). Interestingly, we found that high mRNA expression levels of integrin β3 significantly correlate with worse overall survival in a cohort of TCL patients (p<0.05). Additionally, Ruxolitinib (Ruxo) diminished THs-induced STATs phosphorylation (p<0.05). As Ruxo reported toxic effects we aim to study alternative therapies such as Bexarotene (Bex), a synthetic retinoid used for cutaneous LCT treatment. We found that Bex significantly reduced cell viability in vitro of all TCL subtypes analyzed and combination with Cile resulted in improved anti-lymphoma activity (p<0.01). Also, Ruxo significantly decreased TCL cell viability but to a lesser extent than Bex+Cile (p<0.05). We previously found that BexT4+Cile (T4+, thyroxine supplementation) significantly decreases the in vivo growth of TCL tumors. To understand the mechanisms under this effects we analyzed tumor proteomics profiles and metalloprotease activity after 12 days of treatment. We found that BexT4+Cile regulates proteomic profiles related not only to lymphoma progression (JAK/STAT and PI3K-Akt signaling) but also pathways that impact the tumor microenvironment (TNF and angiogenic factors secretion and Th1/Th2 T cell differentiation). Regarding metalloprotease activity, we found that BexT+Cile inhibited MMP2 and MMP9 activity (p<0.05). Finally, we evaluated the dissemination of TCL in an in vivo model (by vain tail) and found that BexT4+Cile reduced EL4 cell implantation into the liver and kidneys (p<0.05). These results described the mechanisms by which THs induce the activation of JAK/STAT oncogenic pathway and showed how the inhibition of integrin αvβ3 in combination with bexarotene has therapeutic potential for TCL. Citation Format: María M. Debernardi, María V. Revuelta, Helena Sterle, Gonzalo Gonzalez, Ingrid L M Souza, Alejandro Correa, Leandro Cerchietti, Graciela Cremaschi, Florencia Cayrol. Integrin αVβ3 activation by thyroid hormones triggers JAK/STAT oncogenic pathways that promote T cell lymphoma dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 633.

Abstract 7198: A small molecule Notch1 inhibitor (ASR490) restores chemosensitivity in triple-negative breast cancer

Abstract Metastatic triple-negative Breast cancer (mTNBC) is an aggressive and often a chemo resistant disease for which no effective druggable target has yet been identified. We and others reported that the aberrant expression of neurogenic locus Notch homolog protein 1 (Notch1) serves as a molecular signature for TNBC. Hence, we postulated that pharmacologically targeting Notch1 in conjunction with a standard chemotherapeutic agent (Doxorubicin: DXR) may completely eradicate TNBC growth and overcome systemic and debilitating toxicity. Hence, this study aims to elucidate the role of our recently discovered notch1 inhibitor ASR490 and its ability to chemosensitize TNBC without significant systemic toxicity. In our results, pretreatment of 1/10 th of the inhibitory concentration of ASR490 combined with 1/10 th or 1/20th of DXR significantly inhibited the growth of TNBC cells (MDA-MB-231 and BT549). Notably, DXR treatment induced Notch1 activation which led to chemoresistance in TNBC; however, co-treatment with the Notch1 inhibitor ASR490, led to the downregulation of Notch1 activation and eradication of the cell viability and clonogenicity of the TNBC cells. Further, molecular analysis suggested that pretreatment of 1/10th of ASR490 facilitates DXR-mediated sensitivity, by inhibiting the DNA repair pathway and upregulating (cleaved) caspase 3, (cleaved) caspase-9 and poly (ADP-Ribose) polymerase (PARP)-mediated apoptosis. Similarly, the combination effectively blocked the epithelial and mesenchymal transition (EMT) regulators and suppressed metastatic properties of MDA-MB231 and BT-549 cells. Moreover, combining ASR490(1/10th) and DXR (1/20th) significantly eradicated xenotransplanted TNBC tumors compared to ASR490 or DXR alone. In addition to being effective at inhibiting tumor growth, the significantly lower dose requirements of DXR and ASR490 in combination makes it a highly safe therapeutic modality. These findings suggest that aberrant activation of Notch1 is responsible for TNBC chemoresistance and concomitantly inhibiting Notch1 activation is an effective therapeutic strategy to restore chemosensitivity in TNBC. Citation Format: Neha Tyagi, Balaji Chandrasekaran, Ashish Tyagi, Balpreet Singh, Megha Chandran, Amandeep Singh, Arun Sharma, Chendil Damodaran. A small molecule Notch1 inhibitor (ASR490) restores chemosensitivity in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7198.

Abstract 7265: ETS-005, a highly selective TEAD4 palmitoylation inhibitor with potent anti-tumor activity and brain penetrating capability

Abstract The Hippo pathway is a highly conserved network that regulates cell proliferation, tissue regeneration, and organ size by orchestrating YAP/TAZ-TEADs, the core effectors of signaling. Aberrant activation of YAP/TAZ has been implicated in tumorigenesis, tumor progression, metastasis and the resistance to targeted therapeutics, including MAPK and RTK inhibitors. Among the four TEAD paralogs, TEAD4 is found widely overexpressed in various solid tumors and potentially less associated with YAP/TEAD-related renal toxicity. Here we reported the identification of ETS-005, a highly selective TEAD4 small molecule inhibitor that shows preference for binding to the palmitoylation pocket of TEAD4 than other TEAD paralogs over 15 folds. ETS-005 displayed low nanomolar inhibition of cell proliferation in NF2 or LATS1/2 mutant mesothelioma in vitro, and robust anti-tumor efficacy in vivo. ETS-005 showed favorable oral availability, high brain penetration capability and broad safety window. Moreover, ETS-005 had relative long projected T1/2 in human, supporting a more convenient clinical QD dosing schedule. Our study demonstrated that selective TEAD4 palmitoylation inhibitor ETS-005 exhibited potent anti-tumor activity as much as pan-TEAD palmitoylation inhibitors while may gain broader therapeutic index. Additionally, ETS-005 also exhibited profound synergistic effects in drug combination studies, offering broad opportunities combined with MAPK and RTK therapies. ETS-005 is currently in IND-enabling study and expected to enter Phase 1 in 2024. Citation Format: Jiajun Lu, Wenpei Du, Ming Gao, Xiaofei Fan, Jinping Li, Lijian Feng, Mengya Wang, Yingjie Li, Xiaofeng Zhong, Wenqi Cui, Qiangang Zheng, Jidong Zhu. ETS-005, a highly selective TEAD4 palmitoylation inhibitor with potent anti-tumor activity and brain penetrating capability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7265.

Abstract 2026: Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib

Abstract Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, a novel orally active antitumor agent, inhibits the interaction between β-catenin and CREB-binding protein (CBP) and thereby modulates Wnt/β-catenin signaling. We previously reported that E7386 in combination with lenvatinib (LEN), a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs, showed a greater antitumor activity and reduction of tumor microvessels than each treatment alone in preclinical tumor models. Multiple clinical studies are in progress including the combination of E7386 plus LEN (NCT04008797). Here, we further tried to elucidate the mechanism underlying the antitumor and antiangiogenic effect of the combination of E7386 plus LEN by using single-cell RNA sequencing (scRNA-seq) of tumor endothelial cells (ECs). Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma cell line) tumors were treated with E7386 at 25 mg/kg and/or with LEN at 10 mg/kg by daily oral gavage for 8 days. Tumor ECs (CD31+/CD45−) were isolated by magnetic cell separation and flow cytometry and were subjected to scRNA-seq using 10x Genomics pipeline. Bioinformatics data analysis was conducted by Clarivate Analytics. After quality control and filtering, ECs were gated by their expression of pan-EC markers and were further classified into 9 subtypes (tip, capillary, arterial, venous, proliferating, immature, interferon, activated artery, and lymphatic) according to previously reported markers for each subtype. Results: Among classified subtypes, proliferating and immature subtypes showed clear reduction in the percentage of total ECs with combination of E7386 plus LEN versus LEN monotherapy. In both proliferating and immature subtypes, a subset of Wnt/β-catenin downstream genes showed increased expression with LEN monotreatment. Conversely, these Wnt/β-catenin downstream genes were downregulated with the combination of E7386 plus LEN, suggesting that Wnt/β-catenin signaling may be associated with a drug resistance mechanism against LEN in these subtypes. Pathway analysis indicated that genes involved in cell proliferation and survival were downregulated by the E7386 plus LEN combination versus LEN monotreatment in both proliferating and immature subtypes. Conclusion: Taken together, these results suggest a role for E7386 in suppressing the activation of Wnt/β-catenin signaling induced by LEN monotreatment in specific EC subtypes; this activity leads to enhanced antiangiogenic and antitumor activity with E7386 plus LEN combination therapy in a preclinical tumor model. Citation Format: Masahiko Kume, Ryoga Ishida, Yoichi Ozawa, Jialing Shen, Hiroto Konishi, Megumi Kuronishi, Takayuki Kimura, Yusuke Adachi, Yu Kato. Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2026.

Abstract 4364: Molecular insight of non-canonical Gsk3β-Gli-AR axis: Therapeutic implications of truncated-Gli3 in castration-resistant prostate cancer

Abstract The aberrant activation of the hedgehog (Hh) signaling pathway causes the progression of therapy-resistant castration-resistant prostate cancer (CRPC). In this study, we aimed to unravel the biological significance of Gli-family proteins and their functional involvement of Gsk3β-mediated regulatory mechanism(s) in CRPC. We have discovered a novel function of the truncated form of Gli (t-Gli3) in advancing CRPC. This was accomplished through in silico analysis, in vitro experiments, PCa patient clinical data, and in vivo PCa MYC overexpression mouse models. Our investigation into the Gli3 regulation, typically influenced by receptor-mediated ciliary localization, uncovered a Smo-independent mechanism. Surprisingly, despite the lack of a transactivation domain in t-Gli3, we found a specific requirement for an androgen receptor variant 7 (AR-V7) for its action, as evidenced by co-immunoprecipitation and chromatin immunoprecipitation assays. Mechanistically, Gsk3β activation leads to the t-Gli3 generation, and inhibition of Gsk3β supported the accumulation of full-length Gli3 through a non-canonical mechanism. Knockdown of Gsk3β (Gsk3β KD) reduces CRPC cell proliferation in 2-D and 3-D culture models, inhibiting key proliferative signaling pathways and inducing apoptosis via mitochondrial fragmentation. These findings were supported using an orthotropic PCa mouse model. RNA-seq analysis also showed upregulated phenotype enrichment of the apoptosis, P53, and glycolysis pathway genes in 22Rv1Gsk3β KD cells. Compared to scramble, a lower OCR/mitochondrial respiratory rate is evident in 22Rv1Gsk3β KD cells. Next, our exploration of potential therapies for PCa showed that individually targeting Smo or Gli1 is insufficient to inhibit cell viability. Interestingly, we uncovered that solely targeting Gli3 was sufficient to inhibit the growth of CRPC cells. In conclusion, our study provides the significance of Gli-family proteins and novel insights into the interplay of Gsk3β, t-Gli3, and AR, offering therapeutic implications in CRPC. Citation Format: Jyoti Bala Kaushal, Pratima Raut, Sarathy Seshacharyulu, Zahraa W. Alsafwani, Gunjan Sharma, Abdullah M. Khan, Sushanta Halder, Satyanarayana Rachagani, Surinder K. Batra, Jawed A. Siddiqui. Molecular insight of non-canonical Gsk3β-Gli-AR axis: Therapeutic implications of truncated-Gli3 in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4364.

Abstract 5874: Phenotypic plasticity of TKI resistance reprograms cytoskeleton organization through cadherin-catenin and integrin pathways

Abstract The acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) restricts its clinical utility. Clinically, acquired resistance has been highly associated with epithelial-mesenchymal transition (EMT) traits. In this study, we characterized shared phenotypic plasticity features of TKI-resistant cells and aimed to find its key driver in three different cell lines harboring mutant EGFR. In vitro resistance cell lines were generated by dose-escalation exposure to osimertinib, a 3rd generation TKI, for more than 2 months. We employed canonical markers (E-cadherin, vimentin, pan-cytokeratin, and N-cadherin) along with additional markers (EpCAM and fibronectin) to define the EMT status of resistant cells. Moreover, cytoskeleton architecture (F-actin and Vimentin) was utilized to clarify the types of cell motility. Transcriptomics analysis illustrated enriched pathways at focal adhesion and adherens junction. Loss of adherens junction-related catenin (α-E-catenin, β-catenin, γ-catenin, p120 catenin) and aberrant activation of focal adhesion-related protein (p-FAK Y397 and Y576/57, p-Src Y416) confirmed the transcriptomics analysis of reduced contact inhibition and cell polarity. The resulting migration mode of resistant cells highly resembles ameboid movement. Mechanistically, we identified that YTHDF3, a metastasis-promoting regulator of mRNA methylation, destabilizes the mRNA of E-cadherin. Depletion of YTHDF3 in resistant cells reversed the cadherin-catenin pathway and distribution of vimentin and f-actin. In conclusion, resistant cells display ameboid-like movement resulting from loss of contact inhibition and aberrant activation of focal adhesion. Mechanistically, we identified that YTHDF3, a metastasis-promoting regulator of mRNA methylation, destabilizes the mRNA of E-cadherin. Depletion of YTHDF3 in resistant cells reversed the cadherin-catenin pathway and distribution of vimentin and f-actin. YTHDF3 serves as an RNA epitranscriptomic regulator that destabilizes CDH1 mRNA, thereby disrupting adherens junction formation and reducing integrin-mediated focal adhesion stabilization. Citation Format: Chun Ma, Jerry Chieh-Yu Chen, Mong-Lien Wang. Phenotypic plasticity of TKI resistance reprograms cytoskeleton organization through cadherin-catenin and integrin pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5874.

Abstract 5711: Anti-tumor effects of fadraciclib, CDK2/9 inhibitor, in biliary tract cancer

Abstract Background: Targeting CDKs has emerged as a significant strategy in the development of new drugs. The CDK4/6 inhibitors have proven their efficacy in several tumor types, while the CDK2 and CDK9 inhibitors are currently undergoing clinical trials. In biliary tract cancer (BTC), gene expressions of CDK2 and CDK9 are elevated compared to normal tissue. CDK9, a transcriptional CDK, regulates RNA polymerase II (RNAP II), leading to enhanced transcription of oncogenes, such as MCL1. Aberrant activation of these CDKs is associated with cancer progression and evading apoptosis in BTC. Notably, MCL1 is frequently amplified in intrahepatic cholangiocarcinoma (16-21%). This finding underscores the potential significance of CDK2 and CDK9 as therapeutic targets in BTC. However, in BTC, targeting CDK2 or CDK9 has not yet been studied. In this study, we aimed to test CDK2/9 targeting and develop possible biomarker strategies in BTC. Method: A total 9 BTC cell lines (SNU245, SNU308, SNU478, SNU869, SNU1196, SNU2670, SNU2773, TFK1, and HUCCT1) were used. Fadraciclib (CDK2/9 dual inhibitor), Olaparib (PARP inhibitor), JQ1 (BRD4 inhibitor) was used. MTT assay, Colony formation assay, Annexin-V assay, and Cell cycle analysis were performed for evaluation of anti-cancer effects. The effect of Fadraciclib on homologous recombination (HR)-mediated DNA damage repair was assessed using focus formation assay and DRGFP assay. Combination of Fadraciclib with Olaparib or JQ1 was evaluated in vitro and in vivo. Result: The anti-tumor effect of Fadraciclib monotherapy was relatively higher in MCL1-High BTC cells (SNU869, SNU2773) compared to MCL-Low cells (SNU245, SNU2670). In MCL1-High cells, Fadraciclib inhibited CDK9, leading to downregulation of RNAP II phosphorylation at Serine 2 and MCL1. Fadraciclib also interfered with HR-mediated DNA damage response by inhibiting transcription of HR-related genes. The combination of Fadraciclib and Olaparib exhibited synergistic anti-tumor effects in vitro in MCL1-High cells and in vivo xenograft model. Conversely, in MCL1-Low cells, Fadraciclib increased BRD4 mRNA expression, leading to the restoration of CDK9-RNAP II activity. This reactivation of RNAP II restored cellular transcription homeostasis, thereby reactivating the transcription of oncogenes such as MCL1. Combination of Fadraciclib and JQ1 showed synergistic effects in vitro in MCL1-Low cells and in vivo xenograft model by inhibiting restored CDK9-RNAP II activity. Conclusion: MCL1-High BTC cells are sensitive to CDK2/9 inhibitor and show synergism between CDK2/9 inhibitor and PARP inhibitor. In MCL1-Low BTC, a combination of a CDK2/9 inhibitor and a BRD4 inhibitor might represent an optimal strategy for new drug development. Citation Format: Jae-Min Kim, Ah-Rong Nam, Kyoung-Seok Oh, Ju-Hee Bang, Yoojin Jeong, Sea Young Choo, Su In Lee, Hyo Jung Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. Anti-tumor effects of fadraciclib, CDK2/9 inhibitor, in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5711.

Bergaptol inhibits glioma cell proliferation and induces apoptosis via STAT3/Bcl-2 pathway.

Glioblastoma (GBM) is the most common primary malignant brain tumour and lacks therapeutic options with significant effects. The aberrant activation of STAT3 is a critical factor in glioma progression via activating multiple signalling pathways that promote glioma. Among them, the antiapoptotic gene Bcl-2 could be upregulated by p-STAT3, which is an important reason for the continuous proliferation of glioma. We previously reported that bergaptol, a natural furanocoumarin widely found in citrus products, exerts antineuroinflammatory effects by inhibiting the overactivation of STAT3. Here, we aimed to evaluate whether bergaptol could promote glioma apoptosis by inhibiting the STAT3/Bcl-2 pathway. This study found that bergaptol inhibited the proliferation and migration of GBM cell lines (U87 and A172) and promoted apoptosis in vitro. We also found that bergaptol significantly inhibited the STAT3/Bcl-2 pathway in GBM cells. U87 cells were implanted intracranially into nude mice to establish a glioma model, and glioma-bearing mice were treated with bergaptol (40 mg/kg). Bergaptol treatment significantly inhibited glioma growth and prolonged the glioma-bearing mice's survival time. In addition, bergaptol administration also significantly inhibited the STAT3/Bcl-2 pathway of tumour tissue in vivo. Overall, we found that bergaptol could effectively play an antiglioma role by inhibiting STAT3/Bcl-2 pathway, suggesting the potential efficacy of bergaptol in treating glioma.

Mechanisms and therapeutic prospect of the JAK-STAT signaling pathway in liver cancer.

Liver cancer (LC) poses a significant global health challenge due to its high incidence and poor prognosis. Current systemic treatment options, such as surgery, chemotherapy, radiofrequency ablation, and immunotherapy, have shown limited effectiveness for advanced LC patients. Moreover, owing to the heterogeneous nature of LC, it is crucial to uncover more in-depth pathogenic mechanisms and develop effective treatments to address the limitations of the existing therapeutic modalities. Increasing evidence has revealed the crucial role of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in the pathogenesis of LC. The specific mechanisms driving the JAK-STAT pathway activation in LC, participate in a variety of malignant biological processes, including cell differentiation, evasion, anti-apoptosis, immune escape, and treatment resistance. Both preclinical and clinical investigations on the JAK-STAT pathway inhibitors have exhibited potential in LC treatment, thereby opening up avenues for the development of more targeted therapeutic strategies for LC. In this study, we provide an overview of the JAK-STAT pathway, delving into the composition, activation, and dynamic interplay within the pathway. Additionally, we focus on the molecular mechanisms driving the aberrant activation of the JAK-STAT pathway in LC. Furthermore, we summarize the latest advancements in targeting the JAK-STAT pathway for LC treatment. The insights presented in this review aim to underscore the necessity of research into the JAK-STAT signaling pathway as a promising avenue for LC therapy.

Abstract 2022: E7386, a Wnt/β-catenin signaling modulator, suppresses the differentiation of regulatory T cells in combination with lenvatinib plus anti-PD-1 antibody

Abstract Background: E7386, a selective inhibitor of the interaction between β-catenin and CREB binding protein (CBP), showed antitumor activity in preclinical tumor models with aberrant activation of the Wnt/β-catenin signaling pathway. Previously, we reported the enhanced antitumor activity of a triple combination (E7386 in combination with Lenvatinib [LEN, a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs] plus anti-PD-1 antibody) versus a 2-drug combination (LEN plus anti-PD-1 antibody). In this study, we further investigated the immunomodulatory functions of E7386 as a triple combination by single-cell RNA-sequencing (scRNA-Seq) analysis and subsequent functional assays. Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma) tumors were randomized into 4 groups (n=6 in each group), including ⑴ Nontreatment, ⑵ E7386 at 25 mg/kg (PO, QD), ⑶ LEN at 10 mg/kg (PO, QD) plus anti-PD-1 antibody at 10 mg/kg (IP, BIW), and ⑷ triple combination. Tumor tissues were collected after a 2-week treatment period. CD45+ immune cells were isolated from the tumors by magnetic cell separation (MACS) and subjected to scRNA-Seq using 10x Genomics pipelines. Bioinformatics data analysis was conducted with Clarivate Analytics. To evaluate the effects of E7386 on Treg differentiation, an in vitro-induced regulatory T cells (Tregs) differentiation assay was conducted. Naïve CD4+ T cells were isolated by MACS and plated under iTreg differentiation conditions (stimulation of TGF-β and IL-2) with or without treatment of E7386. Induced Tregs and conditional culture medium were collected for evaluation of Treg differentiation, including gene expression and protein analysis. Results: CD45+ immune cells were clustered into 14 cell-type groups. Increased proportion of monocytes, NK cells, Tregs, CD4+ T cells, CD8+ T cells, eosinophils, and neutrophils were observed after LEN plus anti-PD-1 treatment. Among these subpopulations, Treg were most significantly suppressed by E7386 plus LEN plus anti-PD-1. Gene expression analysis revealed that the triple combination suppressed the activated gene expression of the Stat5/Foxp3 axis in CD4+ T cells, particularly Tregs, induced by LEN plus anti-PD-1 treatment. Consistently, the iTreg differentiation assay confirmed the suppressive function of E7386 on the Treg differentiation rate. Multiple subsequent in vitro assays also demonstrated the inhibitory effects of E7386 on TGF-β secretion and the expression levels of TGF-β downstream target genes. Conclusion: In summary, these data suggest a suppressive role of E7386 on Treg differentiation, which leads to antitumor activity of E7386 in combination with lenvatinib and anti-PD-1 antibody in a preclinical tumor model. Citation Format: Jialing Shen, Ryoga Ishida, Masahiko Kume, Takayuki Kimura, Yusuke Adachi, Yu Kato. E7386, a Wnt/β-catenin signaling modulator, suppresses the differentiation of regulatory T cells in combination with lenvatinib plus anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2022.

Abstract 2902: CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Background: Although recent advances in immunotherapy for metastatic melanoma have transformed the treatment paradigm for a disease that once carried a grim prognosis, nearly 50% of patients fail to reach a complete response to immune checkpoint blockade (ICB) due to multiple mechanisms of tumor-induced immune resistance. These challenges are amplified in the CNS tumor microenvironment (TME) where metastatic brain melanoma (MBM) demonstrates diminished clinical benefit to ICB treatment. A deeper understanding of these mechanisms are required to improve therapeutic response to ICB for these patients. Our group has previously identified that aberrant activation of the myddosomal cascade through downstream interleukin (IL)-1 receptor-associated kinase (IRAK-4) signaling results in chronic stimulation of MAPK and NF-κB in MBM. Further investigation reveals myddosome signaling emanates in large part from the TME, resulting in reflexive immune suppression. CA-4948 (emavusertib), a small molecule inhibitor of IRAK-4, is capable of reaching therapeutic dose levels in the brain where it demonstrates on-target inhibition of this pathway. These data highlight a unique therapeutic opportunity where selective inhibition of immune-suppressive signaling via targeted IRAK-4 blockade may sensitize MBM to anti-PD-1 ICB. Methods Multispectral imaging of patient MBM samples were analyzed for myddosome expression in the TME. Melanoma preclinical models, including both primary site (cutaneous) and metastatic brain disease, were evaluated for tumor burden and survival in response to CA-4948 treatment alone and in combination with anti-PD-1 ICB. Immunological responses of tumor to treatment were evaluated in two transgenic reporter models, CCR2RFPCX3CR1GFP and IFNγIRES-EYFP, to determine both the distribution and activation of infiltrating immune cells. Tumor-associated myeloid cells were assessed for phenotypic markers by multiparameter flow cytometry. Results Myddosome signaling was found to be elevated in tumor-associated immune cells in patient MBM specimens. Treatment of preclinical melanoma with CA-4948 combined with anti-PD-1 ICB resulted in significantly reduced tumor growth and improved survival in three independent models. CA-4948 treatment was associated with reduced tumor recruitment of peripherally derived tumor-associated macrophages, as well as increased lymphocyte infiltration and improved pro-inflammatory signaling measured by increased IFNγ expression in the combinatorial setting. Conclusion Targeted inhibition of IRAK-4 with CA-4948 improves immune surveillance and activation in preclinical models when combined with anti-PD-1 ICB, resulting in reduced tumor growth and increased survival. Thus, CA-4948 may be an effective adjuvant treatment strategy alongside anti-PD-1 ICB for the treatment of metastatic melanoma. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Amanda M. Acevedo, Jeet A. Patel, Savannah L. Carpenter, Alisha Bhatia, Reinhard W. von Roemeling, Jamison Hoffman, Maureen Lane, Duane A. Mitchell. CA-4948 (emavusertib) improves treatment response of preclinical metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2902.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1634.

Abstract 5939: Enhancing nab-paclitaxel chemotherapy response in gastric cancer preclinical models through Inhibition of the HGF/c-Met pathway with merestinib

Abstract Background: Current treatments for primary metastatic or recurrent gastric adenocarcinoma (GAC) primarily involve combination chemotherapy, but the median survival time remains under a year. Nab-paclitaxel has displayed significant antitumor activity in preclinical GAC studies. GAC often exhibits overexpression of various growth factors and their receptors, which play a critical role in its pathophysiology. Aberrant activation of the HGF/c-Met pathway has been reported in up to 50% of GAC patients. We investigated the therapeutic potential of merestinib, a novel inhibitor targeting c-Met, along with Axl and DDR1/2 pathways, in combination with nab-paclitaxel in GAC preclinical models. Methods: Animal survival studies were conducted using peritoneal dissemination xenografts in NOD/SCID mice implanted with MKN-45 cells. Tumor growth was assessed in subcutaneous xenografts using MKN-45 and SNU-1 cells in NOD/SCID mice. IHC analyses were performed to evaluate tumor cell proliferation and microvessel density. In vitro cell proliferation and protein expression were assessed using the WST-1 assay and Western blot analysis, respectively. Results: Animal survival significantly improved with nab-paclitaxel (118%) and merestinib (41%) individually. The combination of merestinib with nab-paclitaxel (153%) resulted in further increased animal survival. In MKN-45 xenografts with high c-Met expression, a substantial reduction in tumor growth was observed with nab-paclitaxel and merestinib, showing a synergistic response. The net change in tumor size in the control, nab-paclitaxel, merestinib, and combination groups was 503 mm3, 115 mm3, 91 mm3, and -9.7 mm3 (tumor regression). In low c-Met expressing SNU-1 xenografts, the effect of merestinib and nab-paclitaxel was less pronounced compared to MKN-45 xenografts. The net change in tumor size in the control, nab-paclitaxel, merestinib, and combination groups was 219 mm3, 105 mm3, 131 mm3, and 57 mm3. Tumor cell proliferation and microvessel density corroborated the tumor growth study results. Nab-paclitaxel and merestinib reduced in vitro cell proliferation, with an additive effect in combination. The reduction in cell proliferation by nab-paclitaxel (10 nM), merestinib (100 nM), and their combination was 87%, 82%, and 94% in MKN-45, 59%, 50%, and 82% in SNU-1, and 53%, 19%, and 66% in gastric fibroblasts. In MKN-45 cells, merestinib increased the expression of pro-apoptotic proteins and decreased the expression of phospho-c-Met, phospho-EGFR, phospho-IGF-1R, phospho-ERK, and phospho-AKT. Conclusion:Merestinib has strong antitumor activity in GAC, especially when administered with nab-paclitaxel, demonstrating a synergistic effect. These results provide compelling evidence for the potential clinical relevance of this combination therapy in improving the survival of GAC patients. Citation Format: Quinn Kaurich, Jennifer Huang, Sazzad Hassan, Urs von Holzen, Niranjan Awasthi. Enhancing nab-paclitaxel chemotherapy response in gastric cancer preclinical models through Inhibition of the HGF/c-Met pathway with merestinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5939.

Abstract 3006: Morphogen-driven regulation of nuclear shape and its implication for colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide despite improved rates of early detection and development of targeted treatments. Dysregulation of nuclear shape and the emergence of aberrant chromatin architecture correlate with cancer onset and progression, including CRC. Both extrinsic and intrinsic factors, most critically cytoskeletal and nucleoskeletal organization, chromatin architecture, and metabolic signaling, impact nuclear shape. Yet, what precisely determines nuclear shape in health and disease and how it is linked to cell fate and function remains a fundamental open question. In the case of over 75% of CRC cases, the first transformative genetic event is the loss of function in the tumor suppressor gene, adenomatous polyposis coli (APC). Mechanistically, perturbed APC signaling leads to aberrant activation of Wnt/beta-catenin signaling. This pathway is essential for intestinal homeostasis, as is the regulation of proper nuclear shape and function. Here, we aim to decipher the crosstalk between Wnt gradients and nuclear shape regulation in non-transformed and APC-mutated epithelial cells in vitro, as well as in WT and APC-mutant mouse colon tissues. The long-term goal of this work is to test whether a mechanistic link exists between Wnt-driven cellular transformation and the emergence of aberrant nuclear shape and function. Citation Format: Mona A. El Gendi, Solène Hervé, Sudeep Gautam, Yekaterina A. Miroshnikova. Morphogen-driven regulation of nuclear shape and its implication for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3006.

Abstract 1666: A novel sox-based continuous and homogeneous assay for the discovery of inhibitors of inactive and active AKT

Abstract Introduction: The activity of protein kinases plays a critical role in the aberrant activation of oncogenic signaling pathways which can drive tumorigenesis and malignant transformation in cancer. AKT has been shown to be both upregulated and mutated in cancer cells allowing it to serve as a driver of cancer cell growth and progression. Inhibition of AKT activation and activity are both attractive targets for effective cancer drug discovery. Experimental Procedures: We developed continuous, homogeneous assays for unactive AKTs. A peptide substrate, modified with a sulfonamido-oxine fluorophore (Sox), utilizes chelation-enhanced fluorescence to enable a real-time readout of AKT-driven phosphorylation. First, a subset of 30,000 Sox-containing sequences were evaluated for AKT substrate candidates, selecting for assay robustness and specificity. A physiologically relevant peptide substrate was identified and used to develop a kinetic assay to monitor AKT1 activation and activity. Unactive AKT1 was incubated with DOPS/DOPC and PIP3, which mimics the plasma membrane, allowing the PH domain of AKT to bind, leading to a conformational change that enables full activation of AKT by PDK1 and MK2. Upon assay initiation, active AKT phosphorylates the sensor peptide, and the resulting signal is read in kinetic mode using a fluorescence intensity readout. First derivative plots for each progress curve were generated; the slope of the linear regions of each plot, representing acceleration towards a steady-state, were used to determine relative rates of activation for each AKT. Results: We developed a novel assay for AKT activation and substrate phosphorylation utilizing AQT0076, DOPS/DOPC, PIP3, PDK1, and MK2. With a mix of classical AKT inhibitors and allosteric inhibitors that rely on an inactive “PH-in” conformation, we demonstrated inhibition and quantified inhibitor potency of both active “PH-out” AKT activity and inactive “PH-in” AKT activation via dose-response measurements of steady-state rate and rate acceleration, respectively. Conclusions: A robust, homogeneous assay was developed to simultaneously monitor AKT activation and substrate phosphorylation kinetically over time. Through a continuous assay, we captured both steady-state rates and rate acceleration as a function of inhibitor concentration, allowing for accurate quantitation of both classes of AKT inhibitors in a single experimental format. With some inhibitors, we observed potencies that matched reported literature values, while with others we observed marked differences that may reflect a more physiological context that could translate through to higher efficacy in the clinic. This novel assay format provides a new tool that can be used in drug discovery to generate more effective next generation inhibitors of both AKT activation and subsequent substrate phosphorylation to prevent cancer cell growth and progression. Citation Format: Susan Cornell-Kennon, Daniel Urul, Matthew Hakar, Hayley McMahon, Khanh Huynh, Earl May, Erik Schaefer. A novel sox-based continuous and homogeneous assay for the discovery of inhibitors of inactive and active AKT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1666.

Abstract 1995: DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer

Abstract The 5-year survival rate for metastatic colorectal cancer (CRC) is less than 14%. Hyperactivation of the ERK/MAPK pathway occurs at a high frequency in CRC, due to mutations (RAS, BRAF) or overexpression/amplification (EGFR) of its signalling components. Aberrant activation of this pathway drives tumour growth and survival and several inhibitors of this pathway are now clinically utilized. The BRAF V600E activating mutation occurs in ~10% of CRC patients and is correlated with an extremely poor prognosis. Use of the small molecule BRAF inhibitor encorafenib in combination with the EGFR inhibitor cetuximab is now approved for BRAF V600E mutant patients, however, the objective response rate is only ~20% and the treatment is rarely curative. Therefore, there is an urgent need to identify predictive biomarkers of response to encorafenib+cetuximab treatment. Dual-specificity phosphatase 4 (DUSP4) is rapidly induced upon activation of ERK/MAPK signaling upon which it acts as a critical negative regulator of signaling output by directly de-phosphorylating ERK. We have observed that DUSP4 is highly expressed in BRAF-mutant CRC cell lines and primary tumours, although there is a range of expression. Whether DUSP4 expression status impacts on response to encorafenib+cetuximab therapy is unknown. The aim of this study therefore was to determine the role of DUSP4 on sensitivity of BRAF-mutant CRC cells to encorafenib+cetuximab treatment in vitro. To examine this, we determined the impact of DUSP4 knock-down, knock-out and re-expression on sensitivity of BRAF-mutant CRC cancer cell lines to encorafenib+cetuximab treatment. Screening a panel of BRAF mutant CRC cell lines for sensitivity to encorafenib+cetuximab revealed a subset of resistant cell lines had either low or undetectable DUSP4 protein expression, while all sensitive cell lines had high DUSP4 expression. siRNA-mediated knockdown of DUSP4 in DUSP4 expressing BRAF-mutant CRC cell lines increased pERK and pFRA1, suggesting activation of ERK/MAPK signalling. DUSP4 knockdown also reduced sensitivity of BRAF mutant CRC cell lines to encorafenib+cetuximab compared to control. These findings were further validated using DUSP4 knock-out with CRISPR-Cas9. Conversely, DUSP4 re-expression in BRAF-mutant CRC cell lines lacking DUSP4 expression, decreased pERK and increased sensitivity to encorafenib+cetuximab treatment. These results demonstrate that DUSP4 regulates ERK/MAPK signalling in BRAF mutant CRC cell lines and that loss of DUSP4 promotes resistance to encorafenib plus cetuximab treatment. These findings warrant investigation of DUSP4 expression status as a predictive biomarker of encorafenib+cetuximab response in patient samples. Citation Format: Kristen Needham, Fiona Chionh, Stan Kaczmarczyk, Connor Kearney, Laura Jenkins, Ian Luk, John M. Mariadason. DUSP4 loss enhances resistance to encorafenib plus cetuximab in BRAF mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1995.