Drive Cell Proliferation Research Articles

Transcriptional regulation of KCNA2 coding Kv1.2 by EWS::FLI1: involvement in controlling the YAP/Hippo signalling pathway and cell proliferation

BackgroundEwing sarcoma (ES), the second main pediatric bone sarcoma, is characterised by a chromosomal translocation leading to the formation of fusion proteins like EWS::FLI1. While several studies have shown that potassium channels drive the development of many tumours, limited data exist on ES. This work therefore aimed to study the transcriptional regulation of KCNA2 and define the involvement of the Kv1.2 channel encoded by KCNA2 in a key function of ES development, cell proliferation.MethodsKCNA2 expression in patients and cell lines was measured via bioinformatic analysis (RNA-Seq). The presence of a functional Kv1.2 channel was shown using patch-clamp experiments. Molecular biology approaches were used after EWS::FLI1 silencing to study the transcriptional regulation of KCNA2. Proliferation and cell count assessment were performed using cell biology approaches. KCNA2 silencing (siRNA) and RNA-Seq were performed to identify the signalling pathways involved in the ability of KCNA2 to drive cell proliferation. The regulation of the Hippo signalling pathway by KCNA2 was studied by measuring Hippo/YAP target genes expression, while YAP protein expression was studied with Western-Blot and immunofluorescence approaches.ResultsThis research identified KCNA2 (encoding for a functional Kv1.2 channel) as highly expressed in ES biopsies and cell lines. The results indicated a correlation between KCNA2 expression and patient survival. The data also demonstrated that KCNA2/Kv1.2 is a direct target of EWS::FLI1, and identified the molecular mechanisms by which this chimeric protein regulates KCNA2 gene expression at the transcriptional level. Furthermore, the results indicated that KCNA2 expression and Kv1.2 activity regulate ES cell proliferation and that KCNA2 expression drives the Hippo/YAP signalling pathway. Using the specific Kv1.2 channel inhibitor (κ-Conotoxin), the results suggested that two complementary mechanisms are involved in this process, both dependently and independently of Kv1.2 functional channels at the plasma membrane.ConclusionThis study is the first to describe the involvement of KCNA2 expression and Kv1.2 channel in cancer development. The study also unveiled the involvement of KCNA2 in the regulation of the Hippo/YAP signalling cascade. Thus, this work suggests that KCNA2/Kv1.2 could be a potential therapeutic target in ES.

Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

Prostate cancer (PCa) is the most common malignancy diagnosed in men. The purpose of this study was to report the molecular pathways of Homo sapiens solute carrier family 4 member 4 (SLC4A4) in the progression of PCa. Here, we report our findings from clinical specimens of prostatic acinar adenocarcinoma collected from patients. We found that low-grade prostate cancers have higher SLC4A4 expression. We investigated the role of SLC4A4 and the signaling mechanism underlying its role in modulating the PCa progression. Firstly, we reported the SLC4A4/GSK-3β/β-catenin signaling axis, which regulates the clonogenic potential, invasiveness, and metastasis. In this, we found reduced phosphorylation of GSK at serine 21 of α and serine 9 of the β subunit in shSLC4A4 cells of PCa, which ultimately relieved the activity of GSK-3β. This activated GSK-3β phosphorylates β-catenin at Ser33/37 with a subsequently reduced β-catenin level in PCa cells. Our functional analysis revealed that SLC4A4 knockdown retards tumor growth and lowers invasion and migration potential. Secondly, we investigated the SLC4A4/RB axis, which acts to drive cell proliferation. SLC4A4 knockdown decreases the interaction between these molecules with hypophosphorylation of RB protein and cell cycle arrest. Likewise, transcriptome sequencing using the SLC4A4 knockdown in DU145 cells regulates differentiated expressed genes and multiple metabolic pathways. Our results suggest that SLC4A4 may serve as a potential therapeutic target for prostate cancer patients in the future.

Trop2-Targeted Molecular Imaging in Solid Tumors: Current Advances and Future Outlook.

Trophoblast cell surface antigen 2 (Trop2), a transmembrane glycoprotein, plays a dual role in physiological and pathological processes. In healthy tissues, Trop2 facilitates development and orchestrates intracellular calcium signaling. However, its overexpression in numerous solid tumors shifts its function toward driving cell proliferation and metastasis, thus leading to a poor prognosis. The clinical relevance of Trop2 is underscored by its utility as both a biomarker for diagnostic imaging and a target for therapy. Notably, the U.S. Food and Drug Administration (FDA) has approved sacituzumab govitecan (SG), a novel Trop2-targeted agent, for treating triple-negative breast cancer (TNBC) and refractory urothelial cancer, highlighting the significance of Trop2 in clinical oncology. Molecular imaging, a powerful tool for visualizing and quantifying biological phenomena at the molecular and cellular levels, has emerged as a critical technique for studying Trop2. This approach encompasses various modalities, including optical imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), and targeted antibodies labeled with radioactive isotopes. Incorporating Trop2-targeted molecular imaging into clinical practice is vital for the early detection, prognostic assessment, and treatment planning of a broad spectrum of solid tumors. Our review captures the latest progress in Trop2-targeted molecular imaging, focusing on both diagnostic and therapeutic applications across diverse tumor types, including lung, breast, gastric, pancreatic, prostate, and cervical cancers, as well as salivary gland carcinomas. We critically evaluate the current state by examining the relevant applications, diagnostic accuracy, therapeutic efficacy, and inherent limitations. Finally, we analyze the challenges impeding widespread clinical application and offer insights into strategies for advancing the field, thereby guiding future research endeavors.

TMIC-49. TUMOR-DERIVED SPERMIDINE DRIVES IMMUNE SUPPRESSION IN THE GLIOBLASTOMA MICROENVIRONMENT VIA CD8 T CELL DEPLETION AND FUNCTIONAL ATTENUATION

Abstract A hallmark of glioblastoma (GBM) is an immunosuppressive microenvironment that is partially driven by tumor cell-derived secreted factors, highlighting the need for a more complete understanding of local immune suppressive mechanism. Altered secreted metabolite levels are observed in the GBM microenvironment; specifically, spermidine (a polyamine), which functions to drive cell proliferation, apoptosis, T cell lineage commitment, and myeloid cell-mediated suppression. However, the direct link between GBM cell-derived spermidine and the tumor microenvironment has yet to be determined. In syngeneic mouse GBM models, spermidine levels were increased in the tumor microenvironment and exogenous administration of spermidine accelerated tumor aggressiveness in an immune dependent-manner. Mechanistically, spermidine administration increased apoptosis of CD8+ T-cells and generated an exhausted phenotype. When ornithine decarboxylase (ODC1, the rate limiting enzyme in spermidine synthesis), was knocked down in tumor cells, no direct effect was observed on tumor cell growth. However, there was an increase in time to tumor succumbence and a concomitant increase in CD8+ T-cells and decrease in CD8+ T cell exhaustion. In GBM patients, there was a negative correlation between ODC1 expression and CD8+ T-cells present near the tumor. Additionally, patients with a favorable prognosis had significantly lower intratumoral levels of spermidine compared to patients with a poor prognosis. Taken together, these results demonstrate that spermidine functions as a tumor cell-derived metabolite that drives tumor progression through reducing CD8+T cell number and altering their phenotype. These findings have clinical implications as clinical trials using a polyamine biosynthesis inhibitor alone have not been very effective in GBM patients, suggesting the need for additional combination immunotherapies. Finally, spermidine is also produced at prominent levels by commensal gut microbes as well as absorbed from consumption of foods such as mushrooms; investigation into how these distal spermidine sources impact GBM growth via the gut-brain axis is actively ongoing.

Chimeric protein EWS::FLI1 drives cell proliferation in Ewing Sarcoma via aberrant expression of KCNN1/SK1 and dysregulation of calcium signaling.

Ewing sarcoma (ES) is characterized by EWS::FLI1 or EWS::ERG fusion proteins. Knowing that ion channels are involved in tumorigenesis, this work aimed to study the involvement of the KCNN1 gene, which encodes the SK1 potassium channel, in ES development. Bioinformatics analyses from databases were used to study KCNN1 expression in patients and cell lines. Molecular approaches and in vitro assays were used to study the transcriptional regulation of KCNN1 and its involvement in the regulation of ES cell proliferation. KCNN1 is overexpressed in ES patient biopsies, and its expression is inversely correlated with patient survival. EWS::FLI1, like EWS::ERG, promotes KCNN1 and SK1 expression, binding to GGAA microsatellites near the promoter of KCNN1 isoforms. KCNN1 is involved in the regulation of ES cell proliferation, with its silencing being associated with a slowing of the cell cycle, and its expression modulates membrane potential and therefore calcium flux. These results highlight that KCNN1 is a direct target of EWS::FLI1 and EWS::ERG and demonstrate that KCNN1 is involved in the regulation of intracellular calcium activity and ES cell proliferation, making it a promising therapeutic target in ES.

Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

Collagen plays a critical role in regulating breast cancer progression and therapeutic resistance. An improved understanding of both the features and drivers of tumor-permissive and -restrictive collagen matrices are critical to improve prognostication and develop more effective therapeutic strategies. In this study, using a combination of in vitro, in vivo and bioinformatic experiments, we show that type III collagen (Col3) plays a tumor-restrictive role in human breast cancer. We demonstrate that Col3-deficient, human fibroblasts produce tumor-permissive collagen matrices that drive cell proliferation and suppress apoptosis in non-invasive and invasive breast cancer cell lines. In human triple-negative breast cancer biopsy samples, we demonstrate elevated deposition of Col3 relative to type I collagen (Col1) in non-invasive compared to invasive regions. Similarly, bioinformatics analysis of over 1000 breast cancer patient biopsies from The Cancer Genome Atlas BRCA cohort revealed that patients with higher Col3:Col1 bulk tumor expression had improved overall, disease-free, and progression-free survival relative to those with higher Col1:Col3 expression. Using an established 3D culture model, we show that Col3 increases spheroid formation and induces the formation of lumen-like structures that resemble non-neoplastic mammary acini. Finally, our in vivo study shows co-injection of murine breast cancer cells (4T1) with rhCol3-supplemented hydrogels limits tumor growth and decreases pulmonary metastatic burden compared to controls. Taken together, these data collectively support a tumor-suppressive role for Col3 in human breast cancer and suggest that strategies that increase Col3 may provide a safe and effective therapeutic modality to limit recurrence in breast cancer patients.

Nucleolin malonylation as a nuclear-cytosol signal exchange mechanism to drive cell proliferation in Hepatocarcinoma by enhancing AKT translation

Cancer cells undergo metabolic reprogramming that is intricately linked to malignancy. Protein acylations are especially responsive to metabolic changes, influencing signal transduction pathways and fostering cell proliferation. However, as a novel type of acylations, the involvement of malonylation in cancer remains poorly understood. In this study, we observed a significant reduction in malonyl-CoA levels in hepatocellular carcinoma (HCC), which correlated with a global decrease in malonylation. Subsequent nuclear malonylome analysis unveiled nucleolin (NCL) malonylation, which was notably enhanced in HCC biopsies. we demonstrated that NCL undergoes malonylation at lysine residues 124 and 398. This modification triggers the translocation of NCL from the nucleolus to nucleoplasm and cytoplasm, binding to AKT mRNA, and promoting AKT translation in HCC. Silencing AKT expression markedly attenuated HCC cell proliferation driven by NCL malonylation. These findings collectively highlight nuclear signaling in modulating AKT expression, suggesting NCL malonylation as a novel mechanism through which cancer cells drive cell proliferation.

Hippo effector, Yorkie, is a tumor suppressor in select Drosophila squamous epithelia

Mammalian Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) and Drosophila Yorkie (Yki) are transcription cofactors of the highly conserved Hippo signaling pathway. It has been long assumed that the YAP/TAZ/Yki signaling drives cell proliferation during organ growth. However, its instructive role in regulating developmentally programmed organ growth, if any, remains elusive. Out-of-context gain of YAP/TAZ/Yki signaling often turns oncogenic. Paradoxically, mechanically strained, and differentiated squamous epithelia display developmentally programmed constitutive nuclear YAP/TAZ/Yki signaling. The unknown, therefore, is how a growth-promoting YAP/TAZ/Yki signaling restricts proliferation in differentiated squamous epithelia. Here, we show that reminiscent of a tumor suppressor, Yki negatively regulates the cell growth-promoting PI3K/Akt/TOR signaling in the squamous epithelia of Drosophila tubular organs. Thus, downregulation of Yki signaling in the squamous epithelium of the adult male accessory gland (MAG) up-regulates PI3K/Akt/TOR signaling, inducing cell hypertrophy, exit from their cell cycle arrest, and, finally, culminating in squamous cell carcinoma (SCC). Thus, blocking PI3K/Akt/TOR signaling arrests Yki loss-induced MAG-SCC. Further, MAG-SCCs, like other lethal carcinomas, secrete a cachectin, Impl2-the Drosophila homolog of mammalian IGFBP7-inducing cachexia and shortening the lifespan of adult males. Moreover, in the squamous epithelium of other tubular organs, like the dorsal trunk of larval tracheal airways or adult Malpighian tubules, downregulation of Yki signaling triggers PI3K/Akt/TOR-induced cell hypertrophy. Our results reveal that Yki signaling plays an instructive, antiproliferative role in the squamous epithelia of tubular organs.

Overcoming drug resistance of cancer cells by targeting the FGF1/FGFR1 axis with honokiol or FGF ligand trap.

Chemoresistance of cancer cells, resulting from various mechanisms, is a significant obstacle to the effectiveness of modern cancer therapies. Targeting fibroblast growth factors (FGFs) and their receptors (FGFRs) is becoming crucial, as their high activity significantly contributes to cancer development and progression by driving cell proliferation and activating signaling pathways that enhance drug resistance. We investigated the potential of honokiol and FGF ligand trap in blocking the FGF1/FGFR1 axis to counteract drug resistance. Using PEAQ-ITC, we verified direct interaction of honokiol with the FGFR1 kinase domain. We then demonstrated the effect of FGF1/FGFR1 inhibition on taltobulin resistance in cells expressing FGFR1. Finally, we generated drug-resistant clones by prolonged exposure of cells with negligible FGFR levels to taltobulin alone, taltobulin and honokiol, or taltobulin and FGF ligand trap. We demonstrated for the first time a direct interaction of honokiol with the FGFR1 kinase domain, resulting in inhibition of downstream signaling pathways. We revealed that both honokiol and FGF ligand trap prevent FGF1-dependent protection against taltobulin in cancer cells expressing FGFR1. In addition, we showed that cells obtained by long-term exposure to taltobulin are resistant to both taltobulin and other microtubule-targeting drugs, and exhibit elevated levels of FGFR1 and cyclin D. We also found that the presence of FGF-ligand trap prevents the development of long-term resistance to taltobulin. Our results shed light on how blocking the FGF1/FGFR1 axis by honokiol and FGF ligand trap could help develop more effective cancer therapies, potentially preventing the emergence of drug-resistant relapses.

GNA13 suppresses proliferation of ER+ breast cancer cells via ERα dependent upregulation of the MYC oncogene

GNA13 (Gα13) is one of two alpha subunit members of the G12/13 family of heterotrimeric G-proteins which mediate signaling downstream of GPCRs. It is known to be essential for embryonic development and vasculogenesis and has been increasingly shown to be involved in mediating several steps of cancer progression. Recent studies found that Gα13 can function as an oncogene and contributes to progression and metastasis of multiple tumor types, including ovarian, head and neck and prostate cancers. In most cases, Gα12 and Gα13, as closely related α-subunits in the subfamily, have similar cellular roles. However, in recent years their differences in signaling and function have started to emerge. We previously identified that Gα13 drives invasion of Triple Negative Breast Cancer (TNBC) cells in vitro. As a highly heterogenous disease with various well-defined molecular subtypes (ER+ /Her2−, ER+ /Her2+, Her2+, TNBC) and subtype associated outcomes, the function(s) of Gα13 beyond TNBC should be explored. Here, we report the finding that low expression of GNA13 is predictive of poorer survival in breast cancer, which challenges the conventional idea of Gα12/13 being universal oncogenes in solid tumors. Consistently, we found that Gα13 suppresses the proliferation in multiple ER+ breast cancer cell lines (MCF-7, ZR-75-1 and T47D). Loss of GNA13 expression drives cell proliferation, soft-agar colony formation and in vivo tumor formation in an orthotopic xenograft model. To evaluate the mechanism of Gα13 action, we performed RNA-sequencing analysis on these cell lines and found that loss of GNA13 results in the upregulation of MYC signaling pathways in ER+ breast cancer cells. Simultaneous silencing of MYC reversed the proliferative effect from the loss of GNA13, validating the role of MYC in Gα13 regulation of proliferation. Further, we found Gα13 regulates the expression of MYC, at both the transcript and protein level in an ERα dependent manner. Taken together, our study provides the first evidence for a tumor suppressive role for Gα13 in breast cancer cells and demonstrates for the first time the direct involvement of Gα13 in ER-dependent regulation of MYC signaling. With a few exceptions, elevated Gα13 levels are generally considered to be oncogenic, similar to Gα12. This study demonstrates an unexpected tumor suppressive role for Gα13 in ER+ breast cancer via regulation of MYC, suggesting that Gα13 can have subtype-dependent tumor suppressive roles in breast cancer.

ENL reads histone β-hydroxybutyrylation to modulate gene transcription.

Histone modifications are typically recognized by chromatin-binding protein modules (referred to as 'readers') to mediate fundamental processes such as transcription. Lysine β-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap should be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark, and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation led to the suppressed expression of genes such MYC that drivecell proliferation. Together, our work offered a chemoproteomics approach and identified ENL as a novel histone β-hydroxybutyrylation effector that regulates gene transcription, providing new insight into the regulation mechanism and function of histone Kbhb.

An open-label study to assess the safety and efficacy of naporafenib (ERAS-254) administered with trametinib in previously treated patients with locally advanced unresectable or metastatic solid tumor malignancies with RAS Q61X mutations (SEACRAFT-1).

TPS3178 Background: The rat sarcoma viral oncogene (RAS)/mitogen-activated protein kinase (MAPK) pathway is a key signaling cascade that drives cell proliferation, differentiation, and survival. Inappropriate activation of this pathway drives tumorigenesis in a subset of solid tumors. There are 3 isoforms of RAS (KRAS, NRAS, and HRAS) with the most commonly mutated codons being 12, 13, and 61. While these codon mutations map to the active site of RAS, they appear to have differential impacts on pathway activation. Mutations at codon 61 (RAS Q61X) are generally thought to be the most MAPK pathway-activating relative to mutations at codons 12 and 13 and are therefore thought to be the most oncogenic. Naporafenib is a pan-RAF inhibitor that is being evaluated in combination with trametinib, which inhibits MEK, a downstream node of the RAS/MAPK pathway. Clinical proof of concept (PoC) in patients with NRASm melanoma (of which 80 to 90% have Q61X mutations) and preliminary PoC for patients with RAS Q61X NSCLC have been established for the combination. Preclinical data across tumor types suggest that RAS Q61X is a potential predictive biomarker for the combination of naporafenib with trametinib, a hypothesis being tested in this trial. Methods: SEACRAFT-1 (NCT05907304) is a Phase 1b, open-label study evaluating the clinical activity and safety of the combination of naporafenib and trametinib in pts with locally advanced, unresectable, or metastatic solid tumor malignancies that are not responsive to standard therapies or for which there are no standard therapies. Eligible pts must be ≥18 years (or ≥12 and <18 years in an adolescent substudy) with documented RAS Q61X mutation by an analytically validated assay. Pts must also have ≥1 measurable lesion per RECIST (Response Evaluation Criteria in Solid Tumors, v1.1) and Eastern Cooperative Oncology Group (ECOG) Performance Status ≤2 (or Karnofsky/Lansky Performance Status ≥70 for adolescent pts). Exclusion criteria include prior therapy with an ERK, MEK, RAF, or RAS inhibitor, QTcF >450 ms at baseline, and left ventricular ejection fraction (LVEF) <50% at baseline. A total of up to 115 pts will be enrolled to receive naporafenib 200 mg BID and trametinib 1 mg QD continuous in 28-day cycles until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint is ORR; secondary endpoints include assessment of PFS, DOR, safety, and tolerability. Clinical trial information: NCT05907304 .

MYC phase separation selectively modulates the transcriptome.

Dysregulation and enhanced expression of MYC transcription factors (TFs) including MYC and MYCN contribute to the majority of human cancers. For example, MYCN is amplified up to several hundredfold in high-risk neuroblastoma. The resulting overexpression of N-myc aberrantly activates genes that are not activated at low N-myc levels and drives cell proliferation. Whether increasing N-myc levels simply mediates binding to lower-affinity binding sites in the genome or fundamentally changes the activation process remains unclear. One such activation mechanism that could become important above threshold levels of N-myc is the formation of aberrant transcriptional condensates through phase separation. Phase separation has recently been linked to transcriptional regulation, but the extent to which it contributes to gene activation remains an open question. Here we characterized the phase behavior of N-myc and showed that it can form dynamic condensates that have transcriptional hallmarks. We tested the role of phase separation in N-myc-regulated transcription by using a chemogenetic tool that allowed us to compare non-phase-separated and phase-separated conditions at equivalent N-myc levels, both of which showed a strong impact on gene expression compared to no N-myc expression. Interestingly, we discovered that only a small percentage (<3%) of N-myc-regulated genes is further modulated by phase separation but that these events include the activation of key oncogenes and the repression of tumor suppressors. Indeed, phase separation increases cell proliferation, corroborating the biological effects of the transcriptional changes. However, our results also show that >97% of N-myc-regulated genes are not affected by N-myc phase separation, demonstrating that soluble complexes of TFs with the transcriptional machinery are sufficient to activate transcription.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Abstract LB265: IpY.20: Innovative strategies for post-CDK estrogen receptor (ER)+ metastatic breast cancer (mBC)

Abstract Cyclin-dependent kinases (CDK) 4/6 are key cell cycle regulators that form complexes with cyclin D1 and the master regulator p27Kip1. This ternary complex acts downstream of the ER- and HER2-pathways, which drive cell proliferation. CDK4/6 phosphorylates the Retinoblastoma protein and other substrates, and keeps p27 from inhibiting G1-associated CDK2. Targeted inhibition of CDK4/6 (CDK4/6i) blocks the growth of ER+ and HER2+ BC by dissolving the CDK4/6-cyclin D1 complex and permitting the shuttling of p27 to inhibit CDK2 complex. Approval of CDK 4/6i such as palbociclib, though a game changer for ER+ mBC, resulted in tumor resistance, leaving patients without effective treatments and a 5-yr survival of only 30%. Although CDK2 inhibitors (CDK2i) address compensatory CDK2 activity bypassing CDK4/6i, a key resistance mechanism, they lack selectivity due to CDK homology, resulting in off-target toxicity. To inhibit CDK2/4/6 simultaneously, combinations of CDK4/6i and CDK2i monotherapies were attempted, but the lack of coordination between CDK4/6 and CDK2 inhibition resulted in suboptimal efficacy. Concarlo presents a unique therapeutic rationale for directly targeting the CDK master regulator p27 to simultaneously inhibit CDK2/4/6 in a concerted manner, one that is guided by the role of p27 to turn all of the G1 CDKs ON and OFF during periods of proliferation and quiescence, respectively. This transition is facilitated by BRK (Breast tumor kinase)-mediated phosphorylation of p27. When p27 is Y phosphorylated, it converts CDK4/6 and CDK2 into open, active complexes. An endogenous BRK-spliced variant ALT can competitively bind to p27, block Y phosphorylation and lock CDK2/4/6 in the OFF conformation. Concarlo engineered a shorter variant of ALT (CCL20) and packaged in cationic liposomes for intracellular delivery, creating a novel IpY.20 drug product. As a single agent, IpY.20 inhibits cell proliferation in treatment-naïve and CDK4i-resistant HR+ cells with reduced non-tumor cell activity. In combination, IpY.20 enhances CDK4/6i responses in treatment-naïve HR+ cells, making it a potential front-line mBC therapy. While other CDKi activity is limited to cytostasis, IpY.20 shows dual cytostasis-cytotoxicity, potentially a more durable option. IpY.20 does not cause neutropenia commonly seen with other CDKis, and rescues the CDK4/6i-induced neutropenia in mice, suggesting better clinical tolerability. These findings underscore the significance of coordinated targeting of CDK2/4/6, making IpY.20 a first-in-class product uniquely exploiting p27-mediated CDK2/4/6 regulation. Direct targeting of p27 presents a new treatment paradigm bypassing the challenges of ATP-competitive CDKis, with potential to expand into tumor types with aberrant p27, CDK2/4/6 activity, or RAS/MAPK pathways that increase CDK4/6 activity. Citation Format: Grace Chen, Natalie Dudas, Kate Coleman, Carolina Guido, Natalia Zisman, Krishna Allamneni, Stacy Wister Blain. IpY.20: Innovative strategies for post-CDK estrogen receptor (ER)+ metastatic breast cancer (mBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB265.

Abstract 5900: INCB161734: A novel, potent, and orally bioavailable KRAS G12D selective inhibitor demonstrates antitumor activity in KRAS G12D mutant tumors

Abstract KRAS G12D (G12D) is one of the most frequent oncogenic driver mutations, and is especially common in pancreatic (PDAC) and colorectal (CRC) cancers. Patients with G12D-mutated disease experience poor treatment outcomes, representing a significant unmet medical need. G12D mutation results in constitutively active signaling, including hyperactivation of the ERK and PI3K pathways, which drive cell proliferation and survival. Here we describe INCB161734, a novel, potent, selective, and orally bioavailable small molecule G12D inhibitor that demonstrates in vivo efficacy in G12D-bearing tumor models. INCB161734 binds to both the GDP and GTP forms of the G12D mutant at the switch II pocket with picomolar affinity (KD), and exhibits &amp;gt;80-fold selectivity over wildtype (WT) KRAS. INCB161734 potently inhibits SOS1-dependent GDP/GTP exchange activity in cell-free assays (IC50 &amp;lt;3 nM), and exhibits &amp;gt;40-fold selectivity for G12D versus WT KRAS. Additionally, INCB161734 demonstrates high selectivity for G12D over WT in various cellular assays using G12D mutant versus WT cancer derived cell lines. INCB161734 potently inhibits ERK phosphorylation (a correlate for KRAS activity), with a mean IC50 of 14.3 nM (range: 1.9-45.2 nM) across 7 human and 3 mouse G12D cell lines; mean 21.5% inhibition was observed at 1 μM (maximum tested concentration) across 14 WT cell lines. Likewise, INCB161734 inhibits proliferation of G12D mutant cell lines, with a mean IC50 of 154 nM (range: 8.3-318 nM) across the same 7 human G12D cell lines; &amp;lt;30% inhibition (mean 13%) was observed at 1 μM across the same 14 WT cell lines. Treatment with INCB161734 induces caspase 3/7 cleavage in a PDAC cell line (G12D HPAC), with EC50 &amp;lt;100 nM; caspase 3/7 cleavage in a WT cell line (NCI-H838) was not induced at doses as high as 5 μM. In addition, INCB161734 induced cell cycle arrest (S-phase inhibition) in the G12D HPAC cell line, with IC50 12.8 nM; S-phase inhibition in the WT NCI-H838 cell line only occurred at much higher doses (IC50 &amp;gt;3.3 μM). INCB161734 demonstrates excellent oral bioavailability with good absorption, low clearance, and low metabolic turnover. Orally dosed INCB161734 clearly shows target engagement, generating continuous near-maximal KRAS inhibition in G12D HPAC mouse xenograft tumors for almost the entire dosing interval. INCB161734 is efficacious against multiple types of G12D-mutated tumors and xenografts, resulting in significant tumor growth inhibition, growth arrest and/or regression in multiple PDAC (HPAC, Panc0403, and 2838c3) and CRC (CT26, GP2D, and LS513) mouse tumor models. These preclinical results demonstrate that INCB161734 is a potent, selective, and orally bioavailable KRAS G12D inhibitor, strongly efficacious against KRAS G12D mutant tumors. The potential benefit of INCB161734 for patients with KRAS G12D mutant disease is under investigation in ongoing clinical trials. Citation Format: Matthew R. Farren, Valerie Roman, Alexandra Gallion, Abdellah Allali-Hassani, Alexander Sokolsky, Weixi Kong, Amanda Smith, Hui Wang, Gina Correa, Marc Deller, Leslie B. Epling, Jessica Procak, Guofeng Zhang, Katherine Pecko, Keith Kennedy, Jason Boer, Kerri Kurzeja-Lipinski, Maryanne Covington, Kwang-Jong Chen, Renee Wallower, Jennifer Rocha, Rina Pan, Anthony Perry, Brad Yuska, Xiaozhao Wang, Ricardo Macarron, Sunkyu Kim. INCB161734: A novel, potent, and orally bioavailable KRAS G12D selective inhibitor demonstrates antitumor activity in KRAS G12D mutant 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 5900.

Abstract 1571: Upregulation of phosphoglyceric acid mutase (PGAM)-1 is related to lymph node metastasis in gastric cancer

Abstract Gastric cancer (GC) is the most common malignancy of the digestive system, causing over 750,000 deaths worldwide. Gastric cancer mortality is largely due to its high level of invasiveness and metastasis to distal organs. Thus, it is an urgent priority to identify mechanisms underlying GC metastasis to improve cancer diagnosis develop of effective biological targeting agents. In this study, we demonstrate that phosphoglyceric acid mutase-1 (PGAM1) was preferentially over-expressed in lymph node metastasis (LNM) tissues of GC patients and correlated with poor prognosis. PGAM1 knock down in two human GC cell lines resulted in impaired cell proliferation and increased cell cycle arrest. Further, PGAM knockdown inhibited migration of both GC cell lines, which correlated with decreased expression of MMP2, MMP9 and ICAM-1. In a murine xenograft model, GC cells with a PGAM1 knockdown displayed decreased growth capacity in vivo. Western blot analysis showed that PGAM1 knockdown in SGC7901 and BGC823 decreased the phosphorylation of Src, FAK and Paxillin relative to the parental cell lines. These results indicate that PGAM1 may play an important role in promoting GC lymph node metastasis by driving cell proliferation and activating cytoskeletal reorganization and cell migration through the regulating Src/FAK/Paxillin pathway. Citation Format: Helei Wang, Zhicheng Liu, Lei Wang, Zitian Wang, Christina Jamieson, Nicholas A. Cacalano. Upregulation of phosphoglyceric acid mutase (PGAM)-1 is related to lymph node metastasis in gastric 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 1571.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [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 2852.

Abstract 3176: Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in colorectal cancer

Abstract Otopetrin 3 (OTOP3) stands out as a pivotal regulator of intracellular pH through its control over proton transportation into cells, affecting fundamental cellular processes. Previous studies have shown that OTOP3 is associated with the prognosis of colorectal cancer (CRC) patients. However, the mechanism underlying OTOP3 regulation remains unclear in cancer. Our investigation reveals OTOP3 as a potent oncogene in CRC, with higher OTOP3 levels correlating with diminished overall survival rates in CRC patients. Comparative analysis between cancerous and normal colon cells highlights a pronounced overexpression of OTOP3 in cancer cells. Suppression of OTOP3 expression significantly curtailed CRC cell proliferation, colony formation, migration, and invasion, concurrently inducing apoptosis and cell cycle arrest, ultimately leading to cancer cell death. Moreover, through CHX, FBS inactivation, and MG132 treatment experiments, it was confirmed that OTOP3 regulates c-Myc expression and stability through ubiquitination and has a critical impact on CRC cell proliferation and growth. Additionally, OTOP3's modulation of HK2, PKM2, and LDHA, underscores its role in impacting cancer cell behavior via glycolytic pathways. In summary, the overexpression of OTOP3 in CRC cells drives cell proliferation and growth through the regulation of c-Myc, impacting crucial cellular processes. Additionally, it promotes these processes by influencing glycolysis. These findings provide valuable insights into potential therapeutic targets for CRC treatment, possibly involving OTOP3 and its associated pathways. Citation Format: Hyeung-Jin Jang, Wona Jee, So-Mi Park, Ye-rin Park, Seok Woo Kim, Ji Hoon Jung. Otopetrin 3 promotes cell proliferation and regulates glycolysis via interaction with MYC in 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 3176.