bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from A TGFβ-Dependent Stromal Subset Underlies Immune Checkpoint Inhibitor Efficacy in DNA Mismatch Repair–Deficient/Microsatellite Instability-High Colorectal Cancer
Authors: Endo Eisei , Okayama Hirokazu , Saito Katsuharu , Nakajima Shotaro , Yamada Leo , Ujiie Daisuke , Kase Koji , Fujita Shotaro , Endo Hisahito , Sakamoto Wataru , Saito Motonobu , Saze Zenichiro , Momma Tomoyuki , Ohki Shinji , Mimura Kosaku , Kono Koji
Published: March 2024

Abstract
Patients with DNA mismatch repair–deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer represent a biomarker-defined population with distinct clinicopathologic features who are susceptible to immune checkpoint inhibitors (ICI). However, their survival outcomes vary considerably and nearly half of them exhibit primary resistance to current ICIs, suggesting substantial molecular heterogeneity even among tumors with dMMR/MSI-H. We conducted an extensive analysis of the tumor microenvironment (TME) using multiple transcriptomic, proteomic, and IHC cohorts of colorectal cancer, comprising 222 dMMR/MSI-H and 1440 MMR-proficient/microsatellite stable tumors. We developed a TGFβ-responsive stromal gene signature and then identified a unique poor prognostic subgroup of patients with dMMR/MSI-H colorectal cancers, characterized by the upregulation of transcriptional programs, including the TGFβ-rich active TME, angiogenesis, M2 macrophage polarization, and the extracellular matrix signature predictive of ICI resistance. The TGFβ-dependent stromal subset within dMMR/MSI-H tumors exhibiting poor survival outcomes was further recapitulated by proteomic datasets and IHC for VCAN protein expressed by cancer-associated fibroblasts. Meanwhile, this dMMR/MSI-H stromal subgroup was enriched neither with CD8+ T-cell infiltration nor common genomic alterations, such as mutation density and BRAF mutations, compared with dMMR/MSI-H tumors without TGFβ-dependent stromal activation. In conclusion, this study revealed a novel stromal subgroup of patients with dMMR/MSI-H colorectal cancer, demonstrating a TGFβ-rich tumor-promoting TME and unfavorable survival outcomes.
Implications:
Dual inhibition of immune checkpoints and TGFβ signaling may offer a promising strategy for these patients.
Data from Crenolanib Regulates ERK and AKT/mTOR Signaling Pathways in RAS/BRAF-Mutated Colorectal Cancer Cells and Organoids
Authors: Fujino Shiki , Miyoshi Norikatsu , Ito Aya , Yasui Masayoshi , Ohue Masayuki , Ogino Takayuki , Takahashi Hidekazu , Uemura Mamoru , Matsuda Chu , Mizushima Tsunekazu , Doki Yuichiro , Eguchi Hidetoshi
Published: March 2024

Abstract
Recently developed molecularly targeted therapies such as EGFR inhibitors have notably improved the prognosis of patients with cancer. However, patients with KRAS and BRAF mutations do not currently benefit from these therapies. Here, we aimed to examine potential effects of crenolanib as a new molecularly targeted therapy in colorectal cancer. We used multiple colorectal cancer cell lines to investigate the growth-inhibitory effect of crenolanib and its effect in combination with other cytotoxic agents. Primary cultures of patient-derived organoids (PDO), a model that reflects the heterogeneity of clinical colorectal cancer, were used to further validate the effects of crenolanib. Unlike cetuximab, crenolanib remarkably suppressed ERK and AKT/mTOR pathways in HT29 cells with BRAF mutation and in HCT116 cells with KRAS mutation with corresponding growth-suppressing effects. Additive or synergistic effects were observed in treatments with combination of crenolanib and other cytotoxic drugs. Moreover, crenolanib suppressed the expression of stem cell markers, such as OCT4, NANOG, and SOX2. These observations were substantiated in seven PDOs with KRAS mutation and two PDOs without KRAS/BRAF mutations, with crenolanib suppressing the growth of all PDOs regardless of their KRAS mutation status. Furthermore, crenolanib abrogated PDGF- and TGFβ-induced increase of OCT4-positive cells in PDOs. Together, these findings suggest that crenolanib may have clinical utility for patients with colorectal cancer, especially patients with KRAS/BRAF mutations.
Implications:
These findings indicate that crenolanib can be a useful target agent for patients with colorectal cancer, especially patients with KRAS/BRAF mutations.
Data from Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions
Authors: Kim Jong Hyuk , Megquier Kate , Thomas Rachael , Sarver Aaron L. , Song Jung Min , Kim Yoon Tae , Cheng Nuojin , Schulte Ashley J. , Linden Michael A. , Murugan Paari , Oseth LeAnn , Forster Colleen L. , Elvers Ingegerd , Swofford Ross , Turner-Maier Jason , Karlsson Elinor K. , Breen Matthew , Lindblad-Toh Kerstin , Modiano Jaime F.
Published: March 2024

Abstract
Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy.
Implications:
This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.
Data from Multiorgan Signaling Mobilizes Tumor-Associated Erythroid Cells Expressing Immune Checkpoint Molecules
Authors: Sano Yasuyo , Yoshida Toshimi , Choo Min-Kyung , Jiménez-Andrade Yanek , Hill Kathryn R. , Georgopoulos Katia , Park Jin Mo
Published: March 2024

Abstract
Hematopoietic-derived cells are integral components of the tumor microenvironment and serve as critical mediators of tumor–host interactions. Host cells derived from myeloid and lymphoid lineages perform well-established functions linked to cancer development, progression, and response to therapy. It is unclear whether host erythroid cells also contribute to shaping the path that cancer can take, but emerging evidence points to this possibility. Here, we show that tumor-promoting environmental stress and tumor-induced hemodynamic changes trigger renal erythropoietin production and erythropoietin-dependent expansion of splenic erythroid cell populations in mice. These erythroid cells display molecular features indicative of an immature erythroid phenotype, such as the expression of both CD71 and TER119 and the retention of intact nuclei, and express genes encoding immune checkpoint molecules. Nucleated erythroid cells with similar properties are present in mouse and human tumor tissues. Antibody-mediated erythropoietin blockade reduces tumor-responsive erythroid cell induction and tumor growth. These findings reveal the potential of tumor-induced erythropoietin and erythroid cells as targets for cancer treatment.
Implications
: Our study identifies erythropoietin and erythroid cells as novel players in tumor–host interactions and highlights the involvement of multiorgan signaling events in their induction in response to environmental stress and tumor growth.
Data from p53 Frameshift Mutations Couple Loss-of-Function with Unique Neomorphic Activities
Authors: Tong David R. , Zhou Wen , Katz Chen , Regunath Kausik , Venkatesh Divya , Ihuegbu Chinyere , Manfredi James J. , Laptenko Oleg , Prives Carol
Published: March 2024

Abstract
p53 mutations that result in loss of transcriptional activity are commonly found in numerous types of cancer. While the majority of these are missense mutations that map within the central DNA-binding domain, truncations and/or frameshift mutations can also occur due to various nucleotide substitutions, insertions, or deletions. These changes result in mRNAs containing premature stop codons that are translated into a diverse group of C-terminally truncated proteins. Here we characterized three p53 frameshift mutant proteins expressed from the endogenous TP53 locus in U2OS osteosarcoma and HCT116 colorectal cancer cell lines. These mutants retain intact DNA-binding domains but display altered oligomerization properties. Despite their abnormally high expression levels, they are mostly transcriptionally inactive and unable to initiate a stimuli-induced transcriptional program characteristic of wild-type p53. However, one of these variant p53 proteins, I332fs*14, which resembles naturally expressed TAp53 isoforms β and γ, retains some residual antiproliferative activity and can induce cellular senescence in HCT116 cells. Cells expressing this mutant also display decreased motility in migration assays. Hence, this p53 variant exhibits a combination of loss-of-gain and gain-of-function characteristics, distinguishing it from both wild type p53 and p53 loss.
Implications:
p53 frameshift mutants display a mixture of residual antiproliferative and neomorphic functions that may be differentially exploited for targeted therapy.
Data from Cross-talk between YAP and RAR-RXR Drives Expression of Stemness Genes to Promote 5-FU Resistance and Self-Renewal in Colorectal Cancer Cells
Authors: Bauzone Marjolaine , Souidi Mouloud , Dessein Anne-Frédérique , Wisztorski Maxence , Vincent Audrey , Gimeno Jean-Pascal , Monté Didier , Van Seuningen Isabelle , Gespach Christian , Huet Guillemette
Published: March 2024

Abstract
The mechanisms whereby the Hippo pathway effector YAP regulates cancer cell stemness, plasticity, and chemoresistance are not fully understood. We previously showed that in 5-fluorouracil (5-FU)–resistant colorectal cancer cells, the transcriptional coactivator YAP is differentially regulated at critical transitions connected with reversible quiescence/dormancy to promote metastasis. Here, we found that experimental YAP activation in 5-FU–sensitive and 5-FU–resistant HT29 colorectal cancer cells enhanced nuclear YAP localization and the transcript levels of the retinoic acid (RA) receptors RARα/γ and RAR target genes CYP26A1, ALDH1A3, and LGR5 through RA Response Elements (RARE). In these two cell models, constitutive YAP activation reinforced the expression of the stemness biomarkers and regulators ALDH1A3, LGR5, and OCT4. Conversely, YAP silencing, RAR/RXR inhibition by the pan-RAR antagonist BMS493, and vitamin A depletion downregulated stemness traits and self-renewal. Regarding the mechanisms engaged, proximity-dependent labeling, nuclear YAP pulldown coupled with mass spectrometry, and chromatin immunoprecipitation (ChIP)/re-ChIP experiments revealed: (i) the nuclear colocalization/interaction of YAP with RARγ and RXRs; and (ii) combined genomic co-occupancy of YAP, RARα/γ, and RXRα interactomes at proximal RAREs of LGR5 and ALDH1A3 promoters. Moreover, activation of the YAP/RAR-RXR cross-talk in colorectal cancer cells promoted RAR self-activation loops via vitamin A metabolism, RA, and active RAR ligands generated by ALDH1A3. Together, our data identify YAP as a bona fide RAR-RXR transcriptional coactivator that acts through RARE-activated stemness genes.
Implications:
Targeting the newly identified YAP/RAR-RXR cross-talk implicated in cancer cell stemness maintenance may lead to multitarget combination therapies for patients with colorectal cancer.
Data from RAD51AP1 Loss Attenuates Colorectal Cancer Stem Cell Renewal and Sensitizes to Chemotherapy
Authors: Bridges Allison E. , Ramachandran Sabarish , Tamizhmani Kavin , Parwal Utkarsh , Lester Adrienne , Rajpurohit Pragya , Morera Daley S. , Hasanali Sarrah L. , Arjunan Pachiappan , Jedeja Ravirajsinh N. , Patel Nikhil , Martin Pamela M. , Korkaya Hasan , Singh Nagendra , Manicassamy Santhakumar , Prasad Puttur D. , Lokeshwar Vinata B. , Lokeshwar Bal L. , Ganapathy Vadivel , Thangaraju Muthusamy
Published: March 2024

Abstract
DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)–induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin/+). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment.
Implications:
This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.
Data from Bidirectional Regulatory Cross-Talk between Cell Context and Genomic Aberrations Shapes Breast Tumorigenesis
Authors: Kumar Brijesh , Bhat-Nakshatri Poornima , Maguire Calli , Jacobsen Max , Temm Constance J. , Sandusky George , Nakshatri Harikrishna
Published: March 2024

Abstract
Breast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context–dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple “normal” epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression.
Implications:
Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.
Data from REX-1 Represses RASSF1a and Activates the MEK/ERK Pathway to Promote Tumorigenesis in Prostate Cancer
Authors: Liu Weijing , Xie An , Tu Chunhua , Liu Weipeng
Published: March 2024

Abstract
Epigenetics play an important role in the pathogenesis of prostate cancer; it is urgent to investigate vital transcription factors in methylation regulation with the aim to develop novel treatment strategies targeting prostate cancer. As a member of the zinc finger protein family, REX-1 (reduced expression-1) is a transcription factor that has been reported to be closely linked to the development of several cancers. So far, the expression level and precise function of REX-1 in prostate cancer remain largely unknown. Here, we show that REX-1 was overexpressed in prostate cancer clinical tissues, and its expression level was closely correlated with patient prognosis. REX-1 affected prostate tumor growth in vivo by MEK/ERK phosphorylation. Mechanistic studies indicated that REX-1 recruited DNMT3b (DNA methyltransferase 3b), inhibited the transcription of RASSF1a (RAS association domain family 1a), and further modulated methylation of RASSF1a promoter. Intervention of the REX-1/DNMT3b/RASSF1a axis may shed light on the development of novel therapeutic approaches for prostate cancer treatment.
Implications:
REX1 overexpression recruits DNMT3b and downregulates RASSF1a by promoter methylation, suggesting that epigenetic intervention may contribute to prostate cancer treatment.
Data from SLX4IP Promotes Telomere Maintenance in Androgen Receptor–Independent Castration-Resistant Prostate Cancer through ALT-like Telomeric PML Localization
Authors: Mangosh Tawna L. , Awadallah Wisam N. , Grabowska Magdalena M. , Taylor Derek J.
Published: March 2024

Abstract
In advanced prostate cancer, resistance to androgen deprivation therapy is achieved through numerous mechanisms, including loss of the androgen receptor (AR) allowing for AR-independent growth. Therapeutic options are limited for AR-independent castration-resistant prostate cancer (CRPC), and defining mechanisms critical for survival is of utmost importance for targeting this lethal disease. Our studies focus on identifying telomere maintenance mechanism (TMM) hallmarks adopted by CRPC to promote survival. TMMs are responsible for telomere elongation to instill replicative immortality and prevent senescence, with the two TMM pathways available being telomerase and alternative lengthening of telomeres (ALT). Here, we show that AR-independent CRPC demonstrates an atypical ALT-like phenotype with variable telomerase expression and activity, whereas AR-dependent models lack discernible ALT hallmarks. In addition, AR-independent CRPC cells exhibited elevated levels of SLX4IP, a protein implicated in promoting ALT. SLX4IP overexpression in AR-dependent C4-2B cells promoted an ALT-like phenotype and telomere maintenance. SLX4IP knockdown in AR-independent DU145 and PC-3 cells led to ALT-like hallmark reduction, telomere shortening, and induction of senescence. In PC-3 xenografts, this effect translated to reduced tumor volume. Using an in vitro model of AR-independent progression, loss of AR in AR-dependent C4-2B cells promoted an atypical ALT-like phenotype in an SLX4IP-dependent manner. Insufficient SLX4IP expression diminished ALT-like hallmarks and resulted in accelerated telomere loss and senescence.
Implications:
This study demonstrates a unique reliance of AR-independent CRPC on SLX4IP-mediated ALT-like hallmarks and loss of these hallmarks induces telomere shortening and senescence, thereby impairing replicative immortality.
Data from Breast Tumor Kinase (Brk/PTK6) Mediates Advanced Cancer Phenotypes via SH2-Domain Dependent Activation of RhoA and Aryl Hydrocarbon Receptor (AhR) Signaling
Authors: Dwyer Amy R. , Kerkvliet Carlos Perez , Krutilina Raisa I. , Playa Hilaire C. , Parke Deanna N. , Thomas Warner A. , Smeester Branden A. , Moriarity Branden S. , Seagroves Tiffany N. , Lange Carol A.
Published: March 2024

Abstract
Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of patients with breast cancer; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of triple-negative breast cancer (TNBC) remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e., via knock-out or add-back) had little effect on primary tumor volume, but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in-frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor–stimulated cell motility relative to SH3-del, KM, or wild-type PTK6 controls. Reverse-phase protein array revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6+/AhR+ primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model.
Implications:
The SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC via RhoA and AhR, identified herein as novel therapeutic targets in PTK6+ breast tumors.
Data from Quantitative Proteomics Identifies Secreted Diagnostic Biomarkers as well as Tumor-Dependent Prognostic Targets for Clear Cell Renal Cell Carcinoma
Authors: Senturk Aydanur , Sahin Ayse T. , Armutlu Ayse , Kiremit Murat C. , Acar Omer , Erdem Selcuk , Bagbudar Sidar , Esen Tarik , Tuncbag Nurcan , Ozlu Nurhan
Published: March 2024

Abstract
Clear cell renal cell carcinoma (ccRCC) is the third most common and most malignant urological cancer, with a 5-year survival rate of 10% for patients with advanced tumors. Here, we identified 10,160 unique proteins by in-depth quantitative proteomics, of which 955 proteins were significantly regulated between tumor and normal adjacent tissues. We verified four putatively secreted biomarker candidates, namely, PLOD2, FERMT3, SPARC, and SIRPα, as highly expressed proteins that are not affected by intratumor and intertumor heterogeneity. Moreover, SPARC displayed a significant increase in urine samples of patients with ccRCC, making it a promising marker for the detection of the disease in body fluids. Furthermore, based on molecular expression profiles, we propose a biomarker panel for the robust classification of ccRCC tumors into two main clusters, which significantly differed in patient outcome with an almost three times higher risk of death for cluster 1 tumors compared with cluster 2 tumors. Moreover, among the most significant clustering proteins, 13 were targets of repurposed inhibitory FDA-approved drugs. Our rigorous proteomics approach identified promising diagnostic and tumor-discriminative biomarker candidates which can serve as therapeutic targets for the treatment of ccRCC.
Implications:
Our in-depth quantitative proteomics analysis of ccRCC tissues identifies the putatively secreted protein SPARC as a promising urine biomarker and reveals two molecular tumor phenotypes.
Data from Loss of 9p21 Regulatory Hub Promotes Kidney Cancer Progression by Upregulating HOXB13
Authors: Baietti Maria Francesca , Zhao Peihua , Crowther Jonathan , Sewduth Raj Nayan , De Troyer Linde , Debiec-Rychter Maria , Sablina Anna A.
Published: March 2024

Abstract
Loss of chromosome 9p21 is observed in one-thirds of clear-cell renal cell carcinoma (ccRCC) and is associated with poorer patient survival. Unexpectedly, 9p21 LOH does not lead to decreased expression of the 9p21 tumor suppressor genes, CDKN2A and CDKN2B, suggesting alternative mechanisms of 9p-mediated tumorigenesis. Concordantly, CRISPR-mediated 9p21 deletion promotes growth of immortalized human embryonic kidney epithelial cells independently of the CDKN2A/B pathway inactivation. The 9p21 locus has a highly accessible chromatin structure, suggesting that 9p21 loss might contribute to kidney cancer progression by dysregulating genes distal to the 9p21 locus. We identified several 9p21 regulatory hubs by assessing which of the 9p21-interacting genes are dysregulated in 9p21-deleted kidney cells and ccRCCs. By focusing on the analysis of the homeobox gene 13 (HOXB13) locus, we found that 9p21 loss relieves the HOXB13 locus, decreasing HOXB13 methylation and promoting its expression. Upregulation of HOXB13 facilitates cell growth and is associated with poorer survival of patients with ccRCC.
Implications:
The results of our study propose a novel tumor suppressive mechanism on the basis of coordinated expression of physically associated genes, providing a better understanding of the role of chromosomal deletions in cancer.
Data from RICTOR Amplification Promotes NSCLC Cell Proliferation through Formation and Activation of mTORC2 at the Expense of mTORC1
Authors: Kim Laura C. , Rhee Christopher H. , Chen Jin
Published: March 2024

Abstract
Non–small cell lung cancer (NSCLC) is characterized by genomic alterations, yet a targetable mutation has not been discovered in nearly half of all patients. Recent studies have identified amplification of RICTOR, an mTORC2-specific cofactor, as a novel actionable target in NSCLC. mTORC2 is one of two distinct mTOR complexes to sense environmental cues and regulate a variety of cellular processes, including cell growth, proliferation, and metabolism, all of which promote tumorigenesis when aberrantly regulated. Interestingly, other components of mTORC2 are not coamplified with RICTOR in human lung cancer, raising the question as to whether RICTOR amplification-induced changes are dependent on mTORC2 function. To model RICTOR amplification, we overexpressed Rictor using the Cas9 Synergistic Activation Mediator system. Overexpression of Rictor increased mTORC2 integrity and signaling, but at the expense of mTORC1, suggesting that overexpressed Rictor recruits common components away from mTORC1. Additionally, Rictor overexpression increases the proliferation and growth of NSCLC 3D cultures and tumors in vivo. Conversely, knockout of RICTOR leads to decreased mTORC2 formation and activity, but increased mTORC1 function. Because Rictor has mTOR-dependent and -independent functions, we also knocked out mLST8, a shared mTOR cofactor but is specifically required for mTORC2 function. Inducible loss of mLST8 in RICTOR-amplified NSCLC cells inhibited mTORC2 integrity and signaling, tumor cell proliferation, and tumor growth. Collectively, these data identify a mechanism for Rictor-driven tumor progression and provide further rationale for the development of an mTORC2-specific inhibitor.
Implications:
RICTOR amplification drives NSCLC proliferation through formation of mTORC2, suggesting mTORC2-specific inhibition could be a beneficial therapeutic option.
Data from SynPathy: Predicting Drug Synergy through Drug-Associated Pathways Using Deep Learning
Authors: Tang Yi-Ching , Gottlieb Assaf
Published: March 2024

Abstract
Drug combination therapy has become a promising therapeutic strategy for cancer treatment. While high-throughput drug combination screening is effective for identifying synergistic drug combinations, measuring all possible combinations is impractical due to the vast space of therapeutic agents and cell lines. In this study, we propose a biologically-motivated deep learning approach to identify pathway-level features from drug and cell lines' molecular data for predicting drug synergy and quantifying the interactions in synergistic drug pairs. This method obtained an MSE of 70.6 ± 6.4, significantly surpassing previous approaches while providing potential candidate pathways to explain the prediction. We further demonstrate that drug combinations tend to be more synergistic when their top contributing pathways are closer to each other on a protein interaction network, suggesting a potential strategy for combination therapy with topologically interacting pathways. Our computational approach can thus be utilized both for prescreening of potential drug combinations and for designing new combinations based on proximity of pathways associated with drug targets and cell lines.
Implications:
Our computational framework may be translated in the future to clinical scenarios where synergistic drugs are tailored to the patient and additionally, drug development could benefit from designing drugs that target topologically close pathways.
Data from TWEAK–Fn14–RelB Signaling Cascade Promotes Stem Cell–like Features that Contribute to Post-Chemotherapy Ovarian Cancer Relapse
Authors: Holmberg Ryne , Robinson Mikella , Gilbert Samuel F. , Lujano-Olazaba Omar , Waters Jennifer A. , Kogan Emily , Velasquez Candyd Lace R. , Stevenson Denay , Cruz Luisjesus S. , Alexander Logan J. , Lara Jacqueline , Mu Emily M. , Camillo Jared Rafael , Bitler Benjamin G. , Huxford Tom , House Carrie D.
Published: March 2024

Abstract
Disease recurrence in high-grade serous ovarian cancer may be due to cancer stem–like cells (CSC) that are resistant to chemotherapy and capable of reestablishing heterogeneous tumors. The alternative NF-κB signaling pathway is implicated in this process; however, the mechanism is unknown. Here we show that TNF-like weak inducer of apoptosis (TWEAK) and its receptor, Fn14, are strong inducers of alternative NF-κB signaling and are enriched in ovarian tumors following chemotherapy treatment. We further show that TWEAK enhances spheroid formation ability, asymmetric division capacity, and expression of SOX2 and epithelial-to-mesenchymal transition genes VIM and ZEB1 in ovarian cancer cells, phenotypes that are enhanced when TWEAK is combined with carboplatin. Moreover, TWEAK in combination with chemotherapy induces expression of the CSC marker CD117 in CD117− cells. Blocking the TWEAK–Fn14–RelB signaling cascade with a small-molecule inhibitor of Fn14 prolongs survival following carboplatin chemotherapy in a mouse model of ovarian cancer. These data provide new insights into ovarian cancer CSC biology and highlight a signaling axis that should be explored for therapeutic development.
Implications:
This study identifies a unique mechanism for the induction of ovarian cancer stem cells that may serve as a novel therapeutic target for preventing relapse.
Data from Tumor-Suppressive and Immune-Stimulating Roles of Cholesterol 25-hydroxylase in Pancreatic Cancer Cells
Authors: McBrearty Noreen , Cho Christina , Chen Jinyun , Zahedi Farima , Peck Amy R. , Radaelli Enrico , Assenmacher Charles-Antoine , Pavlak Clarice , Devine Anne , Yu Pengfei , Lu Zhen , Zhang Hongru , Li Jinyang , Pitarresi Jason R. , Astsaturov Igor , Cukierman Edna , Rustgi Anil K. , Stanger Ben Z. , Rui Hallgeir , Fuchs Serge Y.
Published: March 2024

Abstract
Cholesterol dependence is an essential characteristic of pancreatic ductal adenocarcinoma (PDAC). Cholesterol 25-hydroxylase (CH25H) catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol, which is implicated in inhibiting cholesterol biosynthesis and in cholesterol depletion. Here, we show that, within PDAC cells, accumulation of cholesterol was facilitated by the loss of CH25H. Methylation of the CH25H gene and decreased levels of CH25H expression occurred in human pancreatic cancers and was associated with poor prognosis. Knockout of Ch25h in mice accelerated progression of Kras-driven pancreatic intraepithelial neoplasia. Conversely, restoration of CH25H expression in human and mouse PDAC cells decreased their viability under conditions of cholesterol deficit, and decelerated tumor growth in immune competent hosts. Mechanistically, the loss of CH25H promoted autophagy resulting in downregulation of MHC-I and decreased CD8+ T-cell tumor infiltration. Re-expression of CH25H in PDAC cells combined with immune checkpoint inhibitors notably inhibited tumor growth. We discuss additional benefits that PDAC cells might gain from inactivation of CH25H and the potential translational importance of these findings for therapeutic approaches to PDAC.
Implications:
Loss of CH25H by pancreatic cancer cells may stimulate tumor progression and interfere with immunotherapies.
Data from Mitochondrial Dysfunction Is a Driver of SP-2509 Drug Resistance in Ewing Sarcoma
Authors: Tokarsky E. John , Crow Jesse C. , Guenther Lillian M. , Sherman John , Taslim Cenny , Alexe Gabriela , Pishas Kathleen I. , Rask Galen , Justis Blake S. , Kasumova Ana , Stegmaier Kimberly , Lessnick Stephen L. , Theisen Emily R.
Published: March 2024

Abstract
Expression of the fusion oncoprotein EWS/FLI causes Ewing sarcoma, an aggressive pediatric tumor characterized by widespread epigenetic deregulation. These epigenetic changes are targeted by novel lysine-specific demethylase-1 (LSD1) inhibitors, which are currently in early-phase clinical trials. Single-agent–targeted therapy often induces resistance, and successful clinical development requires knowledge of resistance mechanisms, enabling the design of effective combination strategies. Here, we used a genome-scale CRISPR–Cas9 loss-of-function screen to identify genes whose knockout (KO) conferred resistance to the LSD1 inhibitor SP-2509 in Ewing sarcoma cell lines. Multiple genes required for mitochondrial electron transport chain (ETC) complexes III and IV function were hits in our screen. We validated this finding using genetic and chemical approaches, including CRISPR KO, ETC inhibitors, and mitochondrial depletion. Further global transcriptional profiling revealed that altered complex III/IV function disrupted the oncogenic program mediated by EWS/FLI and LSD1 and blunted the transcriptomic response to SP-2509.
Implications:
These findings demonstrate that mitochondrial dysfunction modulates SP-2509 efficacy and suggest that new therapeutic strategies combining LSD1 with agents that prevent mitochondrial dysfunction may benefit patients with this aggressive malignancy.
Data from MYC Promotes Tyrosine Kinase Inhibitor Resistance in ROS1-Fusion-Positive Lung Cancer
Authors: Iyer Sudarshan R. , Odintsov Igor , Schoenfeld Adam J. , Siau Evan , Mattar Marissa S. , de Stanchina Elisa , Khodos Inna , Drilon Alexander , Riely Gregory J. , Ladanyi Marc , Somwar Romel , Davare Monika A.
Published: March 2024

Abstract
Targeted therapy of ROS1-fusion-driven non–small cell lung cancer (NSCLC) has achieved notable clinical success. Despite this, resistance to therapy inevitably poses a significant challenge. MYC amplification was present in ∼19% of lorlatinib-resistant ROS1-driven NSCLC. We hypothesized that MYC overexpression drives ROS1-TKI resistance. Using complementary approaches in multiple models, including a MYC-amplified patient-derived cell line and xenograft (LUAD-0006), we established that MYC overexpression induces broad ROS1-TKI resistance. Pharmacologic inhibition of ROS1 combined with MYC knockdown were essential to completely suppress LUAD-0006 cell proliferation compared with either treatment alone. We interrogated cellular signaling in ROS1-TKI-resistant LUAD-0006 and discovered significant differential regulation of targets associated with cell cycle, apoptosis, and mitochondrial function. Combinatorial treatment of mitochondrial inhibitors with crizotinib revealed inhibitory synergism, suggesting increased reliance on glutamine metabolism and fatty-acid synthesis in chronic ROS1-TKI treated LUAD-0006 cells. In vitro experiments further revealed that CDK4/6 and BET bromodomain inhibitors effectively mitigate ROS1-TKI resistance in MYC-overexpressing cells. Notably, in vivo studies demonstrate that tumor control may be regained by combining ROS1-TKI and CDK4/6 inhibition. Our results contribute to the broader understanding of ROS1-TKI resistance in NSCLC.
Implications:
This study functionally characterizes MYC overexpression as a novel form of therapeutic resistance to ROS1 tyrosine kinase inhibitors in non–small cell lung cancer and proposes rational combination treatment strategies.
Data from Inactivation of p21-Activated Kinase 2 (Pak2) Inhibits the Development of Nf2-Deficient Tumors by Restricting Downstream Hedgehog and Wnt Signaling
Authors: Sementino Eleonora , Kadariya Yuwaraj , Cheung Mitchell , Menges Craig W. , Tan Yinfei , Kukuyan Anna-Mariya , Shrestha Ujjawal , Karchugina Sofiia , Cai Kathy Q. , Peri Suraj , Duncan James S. , Chernoff Jonathan , Testa Joseph R.
Published: March 2024

Abstract
Because loss of the NF2 tumor suppressor gene results in p21-activated kinase (Pak) activation, PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility, we asked if loss of Pak2, a highly expressed group I PAK member, affects the development of malignant mesothelioma in Nf2;Cdkn2a-deficient (NC) mice and the growth properties of NC mesothelioma cells in culture. In vivo, deletion of Pak2 resulted in a markedly decreased incidence and delayed onset of both pleural and peritoneal malignant mesotheliomas in NC mice. In vitro, Pak2 deletion decreased malignant mesothelioma cell viability, migration, clonogenicity, and spheroid formation. RNA-sequencing analysis demonstrated downregulated expression of Hedgehog and Wnt pathway genes in NC;Pak2−/− mesothelioma cells versus NC;Pak2+/+ mesothelioma cells. Targeting of the Hedgehog signaling component Gli1 or its target gene Myc inhibited cell viability and spheroid formation in NC;P+/+ mesothelioma cells. Kinome profiling uncovered kinase changes indicative of EMT in NC;Pak2−/− mesothelioma cells, suggesting that Pak2-deficient malignant mesotheliomas can adapt by reprogramming their kinome in the absence of Pak activity. The identification of such compensatory pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs.
Implications:
We provide evidence supporting a role for PAK inhibitors in treating NF2-deficient tumors. NF2-deficient tumors lacking Pak2 eventually adapt by kinome reprogramming, presenting opportunities for combination therapies to bypass anti-PAK drug resistance.
Data from XIAP and PHB1 Regulate Anoikis through Competitive Binding to TRAF6
Authors: Yang Bo , Lou Chao , Chen Shengkai , Zhang Zhiyuan , Xu Qin
Published: March 2024

Abstract
Anoikis resistance is a prerequisite for circulating tumor cells to survive. However, the mechanism underlying anoikis resistance is poorly understood. In the current study, the effect of TNF receptor–associated factor 6 (TRAF6)-induced NF-kB activation on anoikis susceptibility in tumor cells was evaluated. Differential TRAF6-binding proteins in anoikis-sensitive versus anoikis-resistant tumor cells were screened by LC/MS-MS analysis. The effects of TRAF6-binding proteins on the stability of TRAF6, the activation of NF-kB signaling and anoikis susceptibility in tumor cells were detected. We found that the loss of TRAF6 expression is an important molecular event linked to anoikis. X-linked inhibitor of apoptosis protein (XIAP), an E3 ligase, can bind, ubiquitinate, and degrade TRAF6 and may lead to inactivation of NF-κB signaling and anoikis sensitivity. High expression of prohibitin 1 (PHB1) competes with XIAP for binding to TRAF6 and confers anoikis resistance to tumor cells. PHB1 and TRAF6 knockdown eliminated tumor cells from the circulation in vivo. Significant correlations between elevated PHB1 and TRAF6 expression and distant metastasis were observed in patients with oral cancer. Collectively, we elucidated a novel mechanism governing anoikis. Our data also indicated that TRAF6 and PHB1 are potential therapeutic targets for tumor cells disseminating in the circulation.
Implications:
Our data implicate that PHB1 competes with XIAP for binding to TRAF6 and confers anoikis resistance to tumor cells.
Data from Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non–Small Cell Lung Cancer
Authors: Slusher Aaron L. , Kim Jeongjin J. , Ribick Mark , Pollens-Voigt Jesse , Bankhead Armand , Palmbos Phillip L. , Ludlow Andrew T.
Published: March 2024

Abstract
Splicing of the hTERT gene to produce the full-length (FL) transcript is necessary for telomerase enzyme activity and telomere-dependent cellular immortality in the majority of human tumors, including non–small cell lung cancer (NSCLC) cells. The molecular machinery to splice hTERT to the FL isoform remains mostly unknown. Previously, we reported that an intron 8 cis-element termed “direct repeat 8” (DR8) promotes FL hTERT splicing, telomerase, and telomere length maintenance when bound by NOVA1 and PTBP1 in NSCLC cells. However, some NSCLC cells and patient tumor samples lack NOVA1 expression. This leaves a gap in knowledge about the splicing factors and cis-elements that promote telomerase in the NOVA1-negative context. We report that DR8 regulates FL hTERT splicing in the NOVA1-negative and -positive lung cancer contexts. We identified splicing factor 3b subunit 4 (SF3B4) as an RNA trans-factor whose expression is increased in lung adenocarcinoma (LUAD) tumors compared with adjacent normal tissue and predicts poor LUAD patient survival. In contrast to normal lung epithelial cells, which continued to grow with partial reductions of SF3B4 protein, SF3B4 knockdown reduced hTERT splicing, telomerase activity, telomere length, and cell growth in lung cancer cells. SF3B4 was also demonstrated to bind the DR8 region of hTERT pre-mRNA in both NOVA1-negative and -positive NSCLC cells. These findings provide evidence that DR8 is a critical binding hub for trans-factors to regulate FL hTERT splicing in NSCLC cells. These studies help define mechanisms of gene regulation important to the generation of telomerase activity during carcinogenesis.
Implications:
Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.
Data from Therapeutic Targeting of Stromal-Tumor HGF-MET Signaling in an Organotypic Triple-Negative Breast Tumor Model
Authors: Singh Sunil , Lamichhane Astha , Rafsanjani Nejad Pouria , Heiss Jacob , Baumann Hannah , Gudneppanavar Ravindra , Leipzig Nic D. , Konopka Michael , Luker Gary D. , Tavana Hossein
Published: March 2024

Abstract
The tumor microenvironment (TME) promotes proliferation, drug resistance, and invasiveness of cancer cells. Therapeutic targeting of the TME is an attractive strategy to improve outcomes for patients, particularly in aggressive cancers such as triple-negative breast cancer (TNBC) that have a rich stroma and limited targeted therapies. However, lack of preclinical human tumor models for mechanistic understanding of tumor–stromal interactions has been an impediment to identify effective treatments against the TME. To address this need, we developed a three-dimensional organotypic tumor model to study interactions of patient-derived cancer-associated fibroblasts (CAF) with TNBC cells and explore potential therapy targets. We found that CAFs predominantly secreted hepatocyte growth factor (HGF) and activated MET receptor tyrosine kinase in TNBC cells. This tumor–stromal interaction promoted invasiveness, epithelial-to-mesenchymal transition, and activities of multiple oncogenic pathways in TNBC cells. Importantly, we established that TNBC cells become resistant to monotherapy and demonstrated a design-driven approach to select drug combinations that effectively inhibit prometastatic functions of TNBC cells. Our study also showed that HGF from lung fibroblasts promotes colony formation by TNBC cells, suggesting that blocking HGF-MET signaling potentially could target both primary TNBC tumorigenesis and lung metastasis. Overall, we established the utility of our organotypic tumor model to identify and therapeutically target specific mechanisms of tumor–stromal interactions in TNBC toward the goal of developing targeted therapies against the TME.
Implications:
Leveraging a state-of-the-art organotypic tumor model, we demonstrated that CAFs-mediated HGF-MET signaling drive tumorigenic activities in TNBC and presents a therapeutic target.
Data from Bemcentinib and Gilteritinib Inhibit Cell Growth and Impair the Endo-Lysosomal and Autophagy Systems in an AXL-Independent Manner
Authors: Zdżalik-Bielecka Daria , Kozik Kamila , Poświata Agata , Jastrzębski Kamil , Jakubik Marta , Miączyńska Marta
Published: March 2024

Abstract
AXL, a receptor tyrosine kinase from the TAM (TYRO3 AXL and MER) subfamily, and its ligand growth arrest-specific 6 (GAS6) are implicated in pathogenesis of a wide array of cancers, acquisition of resistance to diverse anticancer therapies and cellular entry of viruses. The continuous development of AXL inhibitors for treatment of patients with cancer and COVID-19 underscores the need to better characterize the cellular effects of AXL targeting.

In the present study, we compared the cellular phenotypes of CRISPR–Cas9-induced depletion of AXL and its pharmacological inhibition with bemcentinib, LDC1267 and gilteritinib. Specifically, we evaluated GAS6–AXL signaling, cell viability and invasion, the endo-lysosomal system and autophagy in glioblastoma cells. We showed that depletion of AXL but not of TYRO3 inhibited GAS6-induced phosphorylation of downstream signaling effectors, AKT and ERK1/2, indicating that AXL is a primary receptor for GAS6. AXL was also specifically required for GAS6-dependent increase in cell viability but was dispensable for viability of cells grown without exogenous addition of GAS6. Furthermore, we revealed that LDC1267 is the most potent and specific inhibitor of AXL activation among the tested compounds. Finally, we found that, in contrast to AXL depletion and its inhibition with LDC1267, cell treatment with bemcentinib and gilteritinib impaired the endo-lysosomal and autophagy systems in an AXL-independent manner.
Implications:
Altogether, our findings are of high clinical importance as we discovered that two clinically advanced AXL inhibitors, bemcentinib and gilteritinib, may display AXL-independent cellular effects and toxicity.