Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for more deaths than any other malignancy. Despite advances in treatment, it remains highly lethal, with 5-year survival rates showing minimal improvement over the past several decades, highlighting a critical unmet clinical need. Macrophage Migration Inhibitory Factor (MIF) is a multifunctional cytokine that contributes to inflammation and cancer, promoting tumor growth, progression, and metastasis through modulation of the tumor microenvironment, stimulation of angiogenesis, and regulation of immune responses. Polymorphisms in the promoter region of MIF, such as high-expression CATT repeats, influence MIF expression and susceptibility to a range of inflammatory, autoimmune, and malignant disorders, yet their role in lung cancer remains largely unexplored. Therapeutic strategies targeting MIF, including small-molecule inhibitors, antibodies, and peptide-based agents, have shown promise in preclinical models, although their clinical translation is still limited. This review discusses the dual role of MIF in inflammation and oncology, summarizes current therapeutic developments, and emphasizes the potential of MIF-targeted interventions in lung cancer. It discusses the significance of genetic predisposition, particularly high-expression MIF alleles, in guiding personalized treatment strategies for lung cancer and identifying patients who may derive benefit from MIF inhibition.

Cytokines are central regulators of inflammation and immune responses within the tumor microenvironment and have been implicated in cancer progression and prognosis. However, the prognostic value of coordinated cytokine-related transcriptional programs across cancer types has not been systematically explored. Pan-cancer transcriptomic and clinical data were analyzed to construct a cytokine-related prognostic signature using least absolute shrinkage and selection operator (LASSO) Cox regression. Patients were stratified into high-risk and low-risk groups based on the derived risk score. Prognostic performance was evaluated in training and test cohorts, and biological relevance was assessed through survival analyses and pathway-level investigations. A 16-gene cytokine-related signature was established that consistently stratified patients into distinct prognostic groups across multiple cancer types. High cytokine-related risk scores were significantly associated with unfavorable survival outcomes and were linked to enhanced cell cycle activity, epithelial-mesenchymal transition, and extracellular matrix remodeling. Integration of the risk score with clinical variables improved individualized survival prediction. Immunohistochemical analyses further confirmed increased protein expression of representative risk-associated genes, including pannexin 1 (PANX1) and FERM domain containing 8 (FRMD8), in multiple tumor tissues compared with corresponding normal tissues. The cytokine-related prognostic signature captures key inflammatory and immune-related programs underlying tumor aggressiveness and provides a robust tool for pan-cancer risk stratification with potential clinical utility.

Advanced-stage ovarian cancer remains a major clinical challenge because of its aggressive behavior and the frequent development of chemoresistance. The nuclear factor erythroid-derived 2-like 2 (NRF2) signaling pathway regulates cellular redox homeostasis. However, its role in ovarian cancer stem-like cells remains unclear. Therefore, we aimed to investigate the effects of NRF2 overexpression on acetaldehyde dehydrogenase (ALDH)+ KURAMOCHI ovarian cancer cells in vitro and in vivo. In particular, we investigated the effects of NRF2 on tumor-associated behaviors, chemoresistance, and signaling pathways. Lentivirus-mediated NRF2 overexpression activated extracellular signal-regulated kinase and AKT signaling. Moreover, it modulated tumor-associated phenotypes, including proliferation, migration, and invasion. NRF2-overexpressing cells exhibited significantly enhanced migratory and invasive capacities, increased resistance to paclitaxel and carboplatin, and reduced apoptosis. Furthermore, the expression of anti-apoptotic proteins was upregulated, and caspase-3 activation was attenuated. In xenograft models, NRF2 overexpression promoted tumor growth and increased the expression of antioxidant and angiogenic factors, including heme oxygenase-1 and vascular endothelial growth factor A. Collectively, these findings demonstrate that NRF2 regulates ovarian cancer aggressiveness and chemoresistance by coordinating stress response signaling, survival pathways, and tumor progression. Therefore, targeting NRF2-mediated signaling represents a promising therapeutic strategy for overcoming drug resistance and improving outcomes in patients with ovarian cancer.

Upregulation of glutaminase enzymatic activity promotes tumour cell proliferation. Its overexpression correlates with poor disease outcome in patients, including those with breast cancer. A selective glutaminase inhibitor, CB-839, which targets cancer cells by blocking glutamine conversion to glutamate, has shown promising preclinical results as a therapeutic target in triple-negative breast cancer treatment. The current study aimed to determine the importance of glutaminase in Oestrogen Receptor positive/luminal breast cancer to potentially identify therapeutic targets to treat this subtype. In vitro studies using luminal breast cancer cells were performed to investigate the effects of siRNA knockdown of glutaminase genes (GLS and GLS2) and inhibition using CB-839 on functional assays. Silencing GLS in luminal breast cancer cells significantly reduced cell proliferation whilst inducing apoptosis. A similar impact on cell proliferation was observed when silencing GLS2 in luminal B cells, but there was no observed effect on cell apoptosis and cell cycle. There was little effect of GLS inhibition using CB-839 in luminal breast cancer. This study demonstrates that glutaminase is necessary for luminal breast cancer growth and survival. Co-targeting GLS and GLS2 might be a novel approach for the treatment of this subclass. Further functional studies to evaluate the underlying molecular mechanisms of this process are warranted.

Deletion of the tumor suppressor gene phosphatase and tensin homolog (PTEN) in hepatocellular carcinoma (HCC) is associated with a poor response to therapy and reduced survival. In mice, the deletion of PTEN in hepatocytes generates steatosis; however, on the background of steatosis not all emerging HCC cells lack PTEN, suggesting that steatosis confers a metabolic liability to proliferating PTEN-deficient hepatocytes. Here, we show that PTEN-deficient HepG2 cells develop terminal stress in the endoplasmic reticulum (ER) and profound apoptosis when exposed to a mixture of oleic and palmitic acids, while control cells do not. Lipidomic analyses before and after the treatment indicate a higher increase in triglycerides in PTEN KO cells, as well as profound differences in phospholipid concentrations. Although the triglyceride content increases, the coalescence into lipid droplets was impaired in the KO cells, together with a reduction in β-oxidation. Xenograft studies showed that PTEN KO HCC tumors progressed faster than did the control tumors when mice were fed with normal chow and slower under a high-fat diet. We suggest that while the health risks of a fatty acid-rich diet to liver function and the increased propensity to develop HCC are prominent, once a PTEN-deficient HCC has been established, it exposes vulnerability to lipid overload that can be exploited through diet and pharmacological interventions.

Genetic variants specific to anatomical subsites of colorectal cancer are known to play a crucial role in its prognosis and treatment. We undertook a haplotype-based genome-wide association study (GWAS) to identify specific genetic risk loci for three sites: cecum, right colorectum, and left colorectum. Six different haplotype GWAS were performed using familial and sporadic colorectal cancer cases with tumors at three different sites. The studies included 2358 CRC cases and 1642 healthy controls. A logistic regression model using PLINK v.1.07 software was employed, and risk loci with a p-value of 5 × 10-8 were considered statistically significant. In total, 29 distinct risk loci were identified in the analyses of familial and sporadic cases of cecal and proximal colon cancer. The results from the analyses of familial and sporadic left-sided colorectal cancer did not meet the strict criteria for significance. Among the loci that were associated with cecal cancer, 14 were familial, and seven were sporadic. Among the other right-sided colon cancer loci, six were familial, and two were sporadic. Coding genes were found at 18 of the 29 loci. Our findings of site-specific genetic risk loci support the growing evidence for divergent pathways in familial and sporadic colorectal cancer across different colorectal sites. The data support a model where the rise in proximal tumors, both familial and sporadic, is influenced by genetic risk to a higher degree than that of distal tumors. These findings are important for understanding colorectal carcinogenesis and could, after future studies, lead to new applications in cancer prevention, treatment, and prognosis.

Tumor suppressor genes, such as RASSF1A and REST, play a central role in regulating cell proliferation. RASSF1A is frequently inactivated in various cancers, being associated with poor prognosis and metastasis. REST loss promotes the activation of genes related to invasion and estrogen sensitivity. We aimed to evaluate the expression of REST and RASSF1A in peripheral blood of breast cancer patients at different treatment stages and to associate the results with clinical and laboratory variables. Peripheral blood samples from breast cancer patients were collected at diagnosis and at 3 and 6 months after the start of chemotherapy; blood samples from healthy women were also collected. Gene expression was quantified by qPCR and associated with clinical variables. REST expression was significantly lower in patients (p < 0.0001), showing a negative correlation with the BIRADS classification and an AUC of 0.72. RASSF1A showed no significant difference between groups but was negatively correlated with heparanase (r = -0.4213; p < 0.0001). No relevant variations in gene expression were observed among the serial collections, nor associations with histological type. Downregulation of REST expression in the peripheral blood of breast cancer patients suggests its potential as an auxiliary biomarker for diagnosis and risk stratification. RASSF1A was correlated with mechanisms associated with tumor progression but did not differentiate patients from controls.

Programmed Death-Ligand 1 (PD-L1) promotes tumor progression through several mechanisms, including its intrinsic effect on breast cancer cell proliferation via the S-Phase Kinase-Associated Protein 2 (SKP2)-p21Cip1/p27Kip1 (SKP2-p21/p27) axis. However, the specific regulatory signaling through which PD-L1 influences the SKP2-p21/p27 axis to drive cell proliferation remains unclear. To investigate how PD-L1 mediates SKP2-dependent proliferation, proteomic analyses, gene-expression manipulation via knockdown or overexpression, Western blotting, quantitative immunofluorescence, colony-forming assays, real-time cell analysis, and Xenograft-derived cells were used. Proteomic data analysis identified several PD-L1 downstream targets as potential candidate regulators of the SKP2-p21/p27 axis and activators of the PI3K/AKT pathway. Candidate screening by gene knockdown, followed by analyses of SKP2, p21, and p27 protein expression, revealed Livin and Galectin-1 as upstream regulators of the SKP2-p21/p27 axis. Moreover, Western blotting and quantitative immunofluorescence in three breast cancer cell lines confirmed that PD-L1 is an upstream regulator of Livin, Galectin-1, and SKP2 protein expression. Mechanistically, Livin and Galectin-1 enhanced AKT phosphorylation (Ser473) to sustain PI3K/AKT pathway activation in a positive feedback loop to upregulate SKP2 expression. Functional assays, including colony-forming assays and real-time cell analyzer, demonstrated that Livin and Galectin-1 are critical for PD-L1-mediated, SKP2-dependent proliferation. These findings were corroborated in vivo using xenograft-derived cells. Overall, these findings delineate a tumor-intrinsic signaling axis in which PD-L1 upregulates Livin and Galectin-1 to sustain PI3K/AKT activity and drive SKP2-dependent cell proliferation. Targeting Livin and/or Galectin-1 may provide a rational strategy to disrupt PD-L1-associated proliferative signaling and improve combinatorial therapeutic approaches in breast cancer.

Triple-negative breast cancer (TNBC) remains highly aggressive and refractory to conventional treatments, underscoring the need for novel combination strategies. Here, we employed 2D and 3D in vitro models, transcriptomic profiling, and in vivo xenograft studies to evaluate the anticancer efficacy of cannabinoids combined with the terpene β-caryophyllene (BC) in resistant TNBC models. Among the tested cannabinoids, cannabichromene (CBC) exhibited the greatest potency, and its combination with BC at sub-toxic concentrations significantly reduced IC50 values, enhanced cytotoxicity in spheroids, and suppressed colony formation and migration. The combination treatment induced pronounced G1 cell cycle arrest and increased apoptotic cell death. Western blot analyses revealed downregulation of PARP, Survivin, mTOR, Vimentin, Glypican-5, and PD-L1, while RNA sequencing demonstrated suppression of proliferative and migratory signaling pathways alongside activation of apoptosis, autophagy, and ferroptosis-related pathways. In vivo, CBC + BC significantly inhibited tumor growth in MDA-MB-231 xenografts, outperforming single-agent treatments. Collectively, these findings demonstrate that BC synergistically enhances cannabinoid activity, yielding superior antiproliferative and anti-migratory effects, and highlight this combination as a promising therapeutic strategy for resistant TNBC.

Male urological cancers, including clear cell renal cell carcinoma (ccRCC), bladder cancer (BC), and prostate cancer (PCa), are characterized by extensive heterogeneity and complex ecosystems, yet the underlying mechanisms remain incompletely understood. In this study, scRNA-seq, scATAC-seq and spatial transcriptomics data are integrated to systematically characterize the features of the tumor microenvironment (TME). We identify tumor cell subclones and elucidate the impact of chromosomal abnormalities on their characteristic functions. We further identify transcription factor regulatory networks by analyzing tumor cell differentiation trajectories. Importantly, we integrate DNA methylation and SNP information to deeply dissect the tumor cell differentiation process, revealing the multilayer regulatory mechanisms of tumor-related genes. Additionally, we reveal the evolution of cellular states across ecotypes to provide a more comprehensive characterization of TME. Finally, we screened potential therapeutic agents targeting the molecular mechanisms underlying tumor cell differentiation (Amivantamab in ccRCC, Levothyroxine in BC, Ouabain in PCa) and signature genes of ecotype. In conclusion, our work establishes a comprehensive framework for tumor assessment and informs the development of precision therapeutic strategies.

Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers for cancer risk assessment, yet prospective evidence for breast cancer (BC) remains limited. We conducted a nested case-control study within a prospective cohort to examine whether pre-diagnostic circulating miRNAs are associated with subsequent BC risk and to explore their potential relevance in prospective population-based settings. Baseline serum from 160 women (80 incident BC cases; 80 matched controls) was analyzed, with a median time to diagnosis of 8.9 years. Eight candidate miRNAs were quantified by droplet digital PCR (ddPCR) and normalized to miR-484. Group differences were evaluated by non-parametric tests, and odds ratios for BC were estimated using logistic regression models adjusted for established risk factors, with Bonferroni correction for multiple testing. Cases and controls were comparable at baseline. Among the candidates, lower circulating miR-181 levels showed a suggestive inverse association with BC risk in fully adjusted models, while lower Let7 levels showed only a non-significant, hypothesis-generating inverse trend that did not survive Bonferroni correction. No other miRNA displayed clear associations with BC risk. These findings, while preliminary, support further large-scale prospective investigations specifically designed to assess predictive performance and external validation. employing standardized pre-analytical and analytical protocols, repeated sampling, and independent replication/external validation to clarify the etiologic relevance and potential risk-prediction value of circulating miRNAs for BC.

Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer deaths, with resistance to targeted therapies posing a major clinical challenge. Drug-tolerant persister (DTP) cells are key contributors to resistance, and targeting them offers new strategies to enhance existing treatments. MicroRNAs (miRNAs), particularly the tumour-suppressive miR-15/107 family, offer promise due to their ability to target multiple oncogenic pathways. This study evaluated a synthetic consensus miRNA mimic, conmiR-15/107, in NSCLC cell line models. Dose-response assays showed robust, dose-dependent growth inhibition in both EGFR-mutant (PC9) and KRAS-mutant (H358 and A549) lung adenocarcinoma cells, but not in the human bronchial epithelial cell line BEAS-2B. When combined with EGFR inhibitors (osimertinib and gefitinib) in PC9 cells, the mimics showed a higher rate of growth inhibition compared with the controls and reduced IC50 values. Similarly, conmiR-15/107 enhanced growth inhibition by the KRAS inhibitors sotorasib and adagrasib in H358 cells. RT-qPCR confirmed downregulation of conmiR-15/107 targets, including MEK1, BCL2 and BRCA1, suggesting a multi-target mechanism of action. Long-term assays showed that the mimics reduced the survival and delayed the proliferation of DTPs in osimertinib-treated PC9 cells as well as sotorasib-treated H358 cells. These findings support conmiR-15/107 as a potential adjunct to targeted therapy, capable of enhancing treatment efficacy and delaying resistance in lung adenocarcinoma.

Soft tissue sarcomas are rare malignant mesenchymal tumors for which accurate diagnosis, prognostic stratification, and therapeutic decision-making remain challenging. Although histopathology and immunohistochemistry are essential diagnostic tools, they frequently fail to capture the molecular complexity underlying tumor aggressiveness and treatment resistance. In this study, we evaluated the utility of RNA-based next-generation sequencing for the molecular characterization of STS and for elucidating transcriptomic mechanisms associated with aggressive tumor behavior. An observational cohort of 24 patients with histologically confirmed soft tissue sarcomas was analyzed, using adipose and skeletal muscle tissue as controls. RNA was extracted from tumor samples, libraries were prepared with a targeted pan-cancer panel, and sequencing was performed on the Illumina platform, followed by bioinformatic analysis using DRAGEN pipelines and DESeq2. RNA-NGS identified a predominance of single-nucleotide polymorphisms and significant differential gene expression, with overexpression of proliferation-related genes (TOP2A, MKI67, BUB1B), extracellular matrix and microenvironment-associated genes (COL11A1, SPP1), and developmental regulators (HOXD13, MELK). Subgroup analysis revealed a distinct transcriptomic profile in leiomyosarcoma, while gene fusion analysis detected clinically relevant alterations. These findings demonstrate that RNA-NGS provides biologically and clinically meaningful insights into the molecular landscape of soft tissue sarcomas and supports its integration into precision medicine-oriented diagnostic workflows.

Dedifferentiation-the acquisition of an early developmental state-is a hallmark of cancer. However, the underlying mechanisms that lead to cancer-associated dedifferentiation are not fully understood. Transposable elements (TEs) are becoming increasingly recognised as important regulators of development and disease. The recruitment of TE sequences has played an important role in placental evolution, and TE-derived genes play critical roles in placental development. Although important biological differences exist between tumours and the placenta, the placenta shares certain features with tumours, including the capacity to invade surrounding tissue and modulate the maternal immune response. In this regard, TEs have been implicated in cancer development, and are documented to contribute to oncogenesis through multiple different mechanisms. Moreover, cancers reacquire an epigenetic landscape, which is reflective of early development, and which corresponds to increased phenotypic plasticity, including facilitating the activation of early developmental genes. Many cancers can repurpose developmental genes, including TE-associated genes, which may contribute to pathways involved in invasion and metastasis. Determining whether TE activation is a consequence of broader epigenetic reprogramming or actively contributes to dedifferentiation will be important for understanding cancer biology and may facilitate improvements in cancer diagnosis and treatment.

The efficacy of immunotherapy in colorectal cancer (CRC) has long been considered to be closely associated with microsatellite instability (MSI) status. Patients with microsatellite stable (MSS) tumors typically exhibit poor responses to PD-1/PD-L1 inhibitors and a poor prognosis, often being categorized as immunologically 'cold' tumors. However, some MSS patients can still achieve favorable therapeutic responses, sometimes even surpassing those of certain MSI patients. Immune-cold and immune-hot tumor phenotypes are largely determined by the abundance, clonal expansion, and functional states of tumor-infiltrating T cells. This suggests that immunotherapy responses are driven by dynamic remodeling of T-cell clonality rather than by MSI status alone. To elucidate the underlying T cell clonal dynamics, integrated single-cell transcriptome (scRNA-seq) and T cell receptor sequencing (scTCR-seq) data analyses from 43 blood and tissue samples of MSI and MSS colorectal cancer patients before and after anti-PD-1 therapy were performed. Using our developed TCR reconstruction pipeline (TORBiT), we systematically analyzed the clonal architecture of the TCR repertoire, inter-tissue migration, and its association with T-cell functional state transitions. From a TCR clonal kinetic perspective, we revealed two distinct modes of immune Coercion that may further affect the immune response: a "high-fluctuation, deep-exhaustion" pattern in MSI tumors and a "high-baseline, strong-suppression" pattern in MSS tumors. These findings provide a novel theoretical foundation and research perspective for understanding the responsiveness and resistance mechanisms to immune checkpoint inhibitors.

PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs initially identified in germline cells as genome guardians that silence transposable elements. Recent studies have expanded this view, revealing that piRNAs and PIWI proteins are broadly expressed in somatic tissues and participate in epigenetic and post-transcriptional gene regulation. This review systematically summarizes piRNA biogenesis and molecular mechanisms, with a focus on their functional diversification from germline to somatic cells. We detail piRNA dysregulation and its association with various human diseases, including cancer, cardiovascular disorders, neurodegenerative diseases, immune dysfunction, and reproductive disorders. By integrating recent findings, this review provides a comprehensive overview of piRNA-mediated regulatory networks and highlights their potential as novel biomarkers and therapeutic targets.

Glial brain tumours, including astrocytoma IDH (Isocitrate Dehydrogenase) mutant and glioblastoma IDH wild-type, are highly malignant brain tumours with poor clinical outcomes. Genomic instability, encompassing microsatellite (MIN) and chromosomal instability (CIN), drives tumour heterogeneity and evolution. In this study, genomic instability was analysed in 85 patients using AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) by comparing tumour and normal tissue (blood) DNA profiles of the same patient. Both types of alterations were present in all analysed samples, contributing almost equally to the total level of genomic instability. The dominant pattern of genomic instability in our cohort was low overall instability, predominantly manifesting as low-degree microsatellite instability. A general decrease in genomic instability was observed with increasing tumour grade. Glioblastoma IDH wild-type was more prevalent in older patients, whereas astrocytoma IDH mutant predominated in younger individuals. Notably, low genomic instability (both MIN and CIN) was associated with poorer survival in patients over 50 years of age. Females, compared to males, exhibited higher MIN in grade 2 tumours and elevated CIN in grade 4 tumours. Our results confirm that genomic instability contributes to tumour progression, MIN being the pivotal factor, and could serve as a prognostic biomarker in malignant gliomas.

Triple-negative breast cancer (TNBC) lacks effective targeted therapies, and the mechanisms by which developmental endothelial locus-1 (EDIL3/Del-1) promotes TNBC remain incompletely defined. We profiled Del-1 and AMPK subunits in TNBC cell lines by RT-PCR and immunoblotting, performed functional assays in CRISPR/Cas9 Del-1 knockout and AMPKβ-manipulated cells, and evaluated AMPKβ in early-stage TNBC tumors using tissue microarrays (TMA) (immunohistochemistry; n = 100) and AMPKβ2 mRNA quantification. Del-1 and AMPKβ were enriched in TNBC cells, most prominently in the mesenchymal stem-like subtype, whereas AMPKα levels were relatively stable. Increased Del-1 and activated AMPKβ enhanced proliferation and invasion, while Del-1 deletion reduced AMPKβ expression and suppressed tumor-promoting phenotypes. Mechanistically, Del-1 increased AMPKβ phosphorylation at serine 108, and a phospho-mimetic AMPKβ mutant further amplified oncogenic effects. In the pilot TMA study, high AMPKβ protein expression showed a trend toward poorer DFS in Kaplan-Meier analysis, while multivariate analysis identified high AMPKβ protein expression as an independent factor associated with poorer DFS in patients with early TNBC. These data support AMPKβ as a key mediator of Del-1-driven signaling and suggest AMPKβ could be a therapeutic target in aggressive TNBC subsets.

Pituitary neuroendocrine tumors (PitNETs) are usually benign intracranial neoplasms that may exhibit invasion of the cavernous sinus, complicating surgery and increasing the risk of recurrence. This study aimed to investigate membranous E-cadherin (mE-cad) expression across PitNET subtypes and transcription factor (TF) lineages, including Pit-1 (pituitary-specific positive transcription factor 1), SF-1 (Steroidogenic Factor 1), and TPIT (T-box pituitary transcription factor), and its association with tumor invasiveness in sixty-nine patients. mE-cad expression was evaluated as the percentage of positive cells (0%, 1-10%, >10%) and by immunoreactive score (IRS). Staining intensity was scored as: 0, no staining; 1, weak; 2, moderate; 3, strong. The proportion of positive cells was scored as: 0, none; 1, <10%; 2, 10-50%; 3, 51-80%; 4, >80%. Mean mE-cad expression was 5.2% in gonadotroph, 3.2% in corticotroph, 0.5% in lactotroph, and 17.5% in plurihormonal PitNETs. By TF lineage, the mean expression was 5.3% for Pit-1, 3.2% for TPIT, and 5.1% for SF-1. Low mE-cad expression (IRS 1-2) was associated with higher odds of cavernous sinus invasion compared with IRS 3-6 (adjusted OR = 6.0, 95% CI 1.08-33.4, p = 0.04), independent of tumor volume (adjusted OR = 4.0, 95% CI 1.50-10.7, p = 0.01). After restricting the analysis to the gonadotroph PitNET group, tumors with an IRS of 1-2 showed significantly higher invasiveness compared with those with an IRS of 3-6 (p = 0.012). These findings suggest that mE-cad may serve as a biomarker of PitNET invasiveness, with expression varying according to TF lineage and tumor subtype.

CRISPR screens are a powerful functional genomics approach for identifying genes that confer sensitivity and resistance to anti-cancer therapies. CFI-402257 (luvixasertib, 2257) is a small molecule inhibitor of threonine tyrosine kinase (TTK), a promising therapeutic target in genomically unstable cancers due to its critical role in establishing the spindle assembly checkpoint (SAC) during mitosis. To inform its ongoing development and evaluation in clinical trials, we sought to use CRISPR activation (i.e., gain of function) screens to identify cellular mechanisms of resistance to 2257 in models of triple-negative breast cancer (TNBC). In vitro screens conducted in two TNBC cell lines nominated ABCG2 as the top resistance-conferring gene in both models. Validation studies assessing clonogenic survival and apoptosis confirmed that ABCG2 overexpression enhanced TNBC resistance to 2257 in vitro, while knockdown enhanced sensitivity. These findings suggest that 2257 is a substrate of ABCG2's drug efflux activity. However, overexpression of ABCG2 failed to confer resistance to 2257 in TNBC xenografts grown in mice and treated with a moderately active dose and schedule. Our results highlight the potential impact of drug transporters in in vitro CRISPR screens and the importance of confirming the relevance of drug response mechanisms identified in cultured cells using in vivo models that recapitulate drug pharmacokinetics and pharmacodynamics.