Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9β that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9β were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

Comment on Cancer Res. 1999 Nov 15;59(22):5830-5.

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations. SIGNIFICANCE: A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST-dependent pathway and innate immune signaling.

Prostate cancer treatment planning is largely dependent upon examination of core-needle biopsies. The microscopic architecture of the prostate glands forms the basis for prognostic grading by pathologists. Interpretation of these convoluted three-dimensional (3D) glandular structures via visual inspection of a limited number of two-dimensional (2D) histology sections is often unreliable, which contributes to the under- and overtreatment of patients. To improve risk assessment and treatment decisions, we have developed a workflow for nondestructive 3D pathology and computational analysis of whole prostate biopsies labeled with a rapid and inexpensive fluorescent analogue of standard hematoxylin and eosin (H&E) staining. This analysis is based on interpretable glandular features and is facilitated by the development of image translation-assisted segmentation in 3D (ITAS3D). ITAS3D is a generalizable deep learning-based strategy that enables tissue microstructures to be volumetrically segmented in an annotation-free and objective (biomarker-based) manner without requiring immunolabeling. As a preliminary demonstration of the translational value of a computational 3D versus a computational 2D pathology approach, we imaged 300 ex vivo biopsies extracted from 50 archived radical prostatectomy specimens, of which, 118 biopsies contained cancer. The 3D glandular features in cancer biopsies were superior to corresponding 2D features for risk stratification of patients with low- to intermediate-risk prostate cancer based on their clinical biochemical recurrence outcomes. The results of this study support the use of computational 3D pathology for guiding the clinical management of prostate cancer. SIGNIFICANCE: An end-to-end pipeline for deep learning-assisted computational 3D histology analysis of whole prostate biopsies shows that nondestructive 3D pathology has the potential to enable superior prognostic stratification of patients with prostate cancer.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Immunosuppressive myeloid cells play a major role in cancer by negatively regulating immune responses, promoting tumor progression, and limiting the efficacy of cancer immunotherapy. Immunosuppression is mediated by various mechanisms dependent upon the type of myeloid cell involved. In recent years, a more universal mechanism of immunosuppressive activity of myeloid cells has emerged: Generation of oxidized lipids. Oxidized lipids accumulate in all types of myeloid cells and are often transferred between cells. In this review, we discuss mechanisms involved in the generation and biological role of myeloid cell-derived oxidized lipids in cancer.

Basal-like breast cancer is the most aggressive breast cancer subtype with the worst prognosis. Despite its high recurrence rate, chemotherapy is the only treatment for basal-like breast cancer, which lacks expression of hormone receptors. In contrast, luminal A tumors express ERα and can undergo endocrine therapy for treatment. Previous studies have tried to develop effective treatments for basal-like patients using various therapeutics but failed due to the complex and dynamic nature of the disease. In this study, we performed a transcriptomic analysis of patients with breast cancer to construct a simplified but essential molecular regulatory network model. Network control analysis identified potential targets and elucidated the underlying mechanisms of reprogramming basal-like cancer cells into luminal A cells. Inhibition of BCL11A and HDAC1/2 effectively drove basal-like cells to transition to luminal A cells and increased ERα expression, leading to increased tamoxifen sensitivity. High expression of BCL11A and HDAC1/2 correlated with poor prognosis in patients with breast cancer. These findings identify mechanisms regulating breast cancer phenotypes and suggest the potential to reprogram basal-like breast cancer cells to enhance their targetability. SIGNIFICANCE: A network model enables investigation of mechanisms regulating the basal-to-luminal transition in breast cancer, identifying BCL11A and HDAC1/2 as optimal targets that can induce basal-like breast cancer reprogramming and endocrine therapy sensitivity.

Clonal hematopoiesis (CH) is a phenomenon caused by expansion of white blood cells descended from a single hematopoietic stem cell. While CH can be associated with leukemia and some solid tumors, the relationship between CH and lung cancer remains largely unknown. To help clarify this relationship, we analyzed whole-exome sequencing (WES) data from 1,958 lung cancer cases and controls. Potential CH mutations were identified by a set of hierarchical filtering criteria in different exonic regions, and the associations between the number of CH mutations and clinical traits were investigated. Family history of lung cancer (FHLC) may exert diverse influences on the accumulation of CH mutations in different age groups. In younger subjects, FHLC was the strongest risk factor for CH mutations. Association analysis of genome-wide genetic variants identified dozens of genetic loci associated with CH mutations, including a candidate SNP rs2298110, which may promote CH by increasing expression of a potential leukemia promoter gene OTUD3. Hundreds of potentially novel CH mutations were identified, and smoking was found to potentially shape the CH mutational signature. Genetic variants and lung cancer risk factors, especially FHLC, correlated with CH. These analyses improve our understanding of the relationship between lung cancer and CH, and future experimental studies will be necessary to corroborate the uncovered correlations. SIGNIFICANCE: Analysis of whole-exome sequencing data uncovers correlations between clonal hematopoiesis and lung cancer risk factors, identifies genetic variants correlated with clonal hematopoiesis, and highlights hundreds of potential novel clonal hematopoiesis mutations.

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to be an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-κB signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease. SIGNIFICANCE: The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity.

Comment on Cancer Res. 2022 Jan 15;82(2):278-291.

Deficiency of the tumor suppressor Merlin causes development of schwannoma, meningioma, and ependymoma tumors, which can occur spontaneously or in the hereditary disease neurofibromatosis type 2 (NF2). Merlin mutations are also relevant in a variety of other tumors. Surgery and radiotherapy are current first-line treatments; however, tumors frequently recur with limited treatment options. Here, we use human Merlin-negative schwannoma and meningioma primary cells to investigate the involvement of the endogenous retrovirus HERV-K in tumor development. HERV-K proteins previously implicated in tumorigenesis were overexpressed in schwannoma and all meningioma grades, and disease-associated CRL4DCAF1 and YAP/TEAD pathways were implicated in this overexpression. In normal Schwann cells, ectopic overexpression of HERV-K Env increased proliferation and upregulated expression of c-Jun and pERK1/2, which are key components of known tumorigenic pathways in schwannoma, JNK/c-Jun, and RAS/RAF/MEK/ERK. Furthermore, FDA-approved retroviral protease inhibitors ritonavir, atazanavir, and lopinavir reduced proliferation of schwannoma and grade I meningioma cells. These results identify HERV-K as a critical regulator of progression in Merlin-deficient tumors and offer potential strategies for therapeutic intervention. SIGNIFICANCE: The endogenous retrovirus HERV-K activates oncogenic signaling pathways and promotes proliferation of Merlin-deficient schwannomas and meningiomas, which can be targeted with antiretroviral drugs and TEAD inhibitors.

Combination therapies consisting of immune checkpoint inhibitors plus anti-VEGF therapy show enhanced antitumor activity and are approved treatments for patients with renal cell carcinoma (RCC). The immunosuppressive roles of VEGF in the tumor microenvironment are well studied, but those of FGF/FGFR signaling remain largely unknown. Lenvatinib is a receptor tyrosine kinase inhibitor that targets both VEGFR and FGFR. Here, we examine the antitumor activity of anti-PD-1 mAb combined with either lenvatinib or axitinib, a VEGFR-selective inhibitor, in RCC. Both combination treatments showed greater antitumor activity and longer survival in mouse models versus either single agent treatment, whereas anti-PD-1 mAb plus lenvatinib had enhanced antitumor activity compared with anti-PD-1 mAb plus axitinib. Flow cytometry analysis showed that lenvatinib decreased the population of tumor-associated macrophages and increased that of IFNγ-positive CD8+ T cells. Activation of FGFR signaling inhibited the IFNγ-stimulated JAK/STAT signaling pathway and decreased expression of its target genes, including B2M, CXCL10, and PD-L1. Furthermore, inhibition of FGFR signaling by lenvatinib restored the tumor response to IFNγ stimulation in mouse and human RCC cell lines. These preclinical results reveal novel roles of tumor FGFR signaling in the regulation of cancer immunity through inhibition of the IFNγ pathway, and the inhibitory activity of lenvatinib against FGFRs likely contributes to the enhanced antitumor activity of combination treatment comprising lenvatinib plus anti-PD-1 mAb. SIGNIFICANCE: FGFR pathway activation inhibits IFNγ signaling in tumor cells, and FGFR inhibition with lenvatinib enhances antitumor immunity and the activity of anti-PD-1 antibodies.

The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome in prostate cancer. Together, these studies reveal new mechanisms by which RB loss induces cancer progression and highlight the importance of understanding the targets of E2F1 function. SIGNIFICANCE: This study identifies that RB loss in prostate cancer drives cooperation between AR and E2F1 as coregulators of transcription, which is linked to the progression of advanced disease.

Comment in Cancer Res. 2022 Jan 15;82(2):197-198.

Androgen receptor (AR) signaling continues to play a dominant role in all stages of prostate cancer, including castration-resistant prostate cancers (CRPC) that have developed resistance to second generation AR antagonists such as enzalutamide. In this study, we identified a long noncoding RNA (lncRNA), NXTAR (LOC105373241) that is located convergent with the AR gene and is repressed in human prostate tumors and cell lines. NXTAR bound upstream of the AR promoter and promoted EZH2 recruitment, causing significant loss of AR (and AR-V7) expression. Paradoxically, AR bound the NXTAR promoter, and inhibition of AR by the ACK1/TNK2 small molecule inhibitor (R)-9b excluded AR from the NXTAR promoter. The histone acetyltransferase GCN5 bound and deposited H3K14 acetylation marks, enhancing NXTAR expression. Application of an oligonucleotide derived from NXTAR exon 5 (NXTAR-N5) suppressed AR/AR-V7 expression and prostate cancer cell proliferation, indicating the translational relevance of the negative regulation of AR. In addition, pharmacologic restoration of NXTAR using (R)-9b abrogated enzalutamide-resistant prostate xenograft tumor growth. Overall, this study uncovers a positive feedback loop, wherein NXTAR acts as a novel prostate tumor-suppressing lncRNA by inhibiting AR/AR-V7 expression, which in turn upregulates NXTAR levels, compromising enzalutamide-resistant prostate cancer. The restoration of NXTAR could serve as a new therapeutic modality for patients who have acquired resistance to second generation AR antagonists. SIGNIFICANCE: This study identifies NXTAR as a tumor suppressive lncRNA that can epigenetically downregulate AR/AR-V7 expression and provides a therapeutic strategy to reinstate NXTAR expression for treating recurrent CRPC.

Metabolic reprogramming by oncogenic signaling is a hallmark of cancer. Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms inducing hyperactivation of Wnt/β-catenin signaling and strategies for targeting this pathway are incompletely understood. In this study, we find nucleoside diphosphate kinase 7 (NME7) to be a positive regulator of Wnt/β-catenin signaling. Upregulation of NME7 positively correlated with the clinical features of HCC. Knockdown of NME7 inhibited HCC growth in vitro and in vivo, whereas overexpression of NME7 cooperated with c-Myc to drive tumorigenesis in a mouse model and to promote the growth of tumor-derived organoids. Mechanistically, NME7 bound and phosphorylated serine 9 of GSK3β to promote β-catenin activation. Furthermore, MTHFD2, the key enzyme in one-carbon metabolism, was a target gene of β-catenin and mediated the effects of NME7. Tumor-derived organoids with NME7 overexpression exhibited increased sensitivity to MTHFD2 inhibition. In addition, expression levels of NME7, β-catenin, and MTHFD2 correlated with each other and with poor prognosis in patients with HCC. Collectively, this study emphasizes the crucial roles of NME7 protein kinase activity in promoting Wnt/β-catenin signaling and one-carbon metabolism, suggesting NME7 and MTHFD2 as potential therapeutic targets for HCC. SIGNIFICANCE: The identification of NME7 as an activator of Wnt/β-catenin signaling and MTHFD2 expression in HCC reveals a mechanism regulating one-carbon metabolism and potential therapeutic strategies for treating this disease.

Heterogeneity is a pervasive feature of cancer, and understanding the sources and regulatory mechanisms underlying heterogeneity could provide key insights to help improve the diagnosis and treatment of cancer. In this review, we discuss the origin of heterogeneity in the phenotype of individual cancer cells. Genotype-phenotype (G-P) maps are widely used in evolutionary biology to represent the complex interactions of genes and the environment that lead to phenotypes that impact fitness. Here, we present the rationale of an extended G-P (eG-P) map with a cone structure in cancer. The eG-P cone is formed by cells that are similar at the genome layer but gradually increase variability in the epigenome, transcriptome, proteome, metabolome, and signalome layers to produce large variability at the phenome layer. Experimental evidence from single-cell-omics analyses supporting the cancer eG-P cone concept is presented, and the impact of epimutations and the interaction of cancer and tumor microenvironmental eG-P cones are integrated with the current understanding of cancer biology. The eG-P cone concept uncovers potential therapeutic strategies to reduce cancer evolution and improve cancer treatment. More methods to study phenotypes in single cells will be the key to better understand cancer cell fitness in tumor biology and therapeutics.

Comment on Cancer Res. 2022 Jan 1;82(1):130-141.

Comment on Cancer Res. 2002 Dec 1;62(23):6997-7000.

Comment on Cancer Res. 2004 Jun 1;64(11):3731-6.

Measuring the selective fitness advantages provided by driver mutations has the potential to facilitate a precise quantitative understanding of cancer evolution. However, accurately measuring the selective advantage of driver mutations has remained a challenge in the field. Early studies reported small selective advantages of drivers, on the order of 1%, whereas newer studies report much larger selective advantages, as high as 1,200%. In this article, we argue that the calculated selective advantages of cancer drivers are dependent on the underlying mathematical model and stage of cancer evolution and that comparisons of numerical values of selective advantage without regard for the underlying model and stage can lead to spurious conclusions.

Lung cancers are the leading cause of cancer-related mortality worldwide, and the majority of lung cancers are non-small cell lung carcinoma (NSCLC). Overexpressed or activated EGFR has been associated with a poor prognosis in NSCLC. We previously identified a circular noncoding RNA, hsa_circ_0000190 (C190), as a negative prognostic biomarker of lung cancer. Here, we attempted to dissect the mechanistic function of C190 and test the potential of C190 as a therapeutic target in NSCLC. C190 was upregulated in both NSCLC clinical samples and cell lines. Activation of the EGFR pathway increased C190 expression through a MAPK/ERK-dependent mechanism. Transient and stable overexpression of C190 induced ERK1/2 phosphorylation, proliferation, and migration in vitro and xenograft tumor growth in vivo. RNA sequencing and Expression2Kinases (X2K) analysis indicated that kinases associated with cell-cycle and global translation are involved in C190-activated networks, including CDKs and p70S6K, which were further validated by immunoblotting. CRISPR/Cas13a-mediated knockdown of C190 decreased proliferation and migration of NSCLC cells in vitro and suppressed tumor growth in vivo. TargetScan and CircInteractome databases predicted that C190 targets CDKs by sponging miR-142-5p. Analysis of clinical lung cancer samples showed that C190, CDK1, and CDK6 expressions were significantly higher in advanced-stage lung cancer than in early-stage lung cancer. In summary, C190 is directly involved in EGFR-MAPK-ERK signaling and may serve as a potential therapeutic target for the treatment of NSCLC. SIGNIFICANCE: The circRNA C190 is identified as a mediator of multiple pro-oncogenic signaling pathways in lung cancer and can be targeted to suppress tumor progression.

Fibroblast growth factor receptor 3 (FGFR3) is frequently activated by mutation or overexpression, and it is a validated therapeutic target in urothelial carcinoma (UC) of the bladder. However, the role and detailed molecular mechanism of FGFR3 in the immune microenvironment of bladder cancer remain largely unknown. Here, we demonstrate that inhibition of FGFR3 in FGFR3-activated bladder cancer elevates PD-L1 protein levels by affecting its ubiquitination, thereby inhibiting the antitumor activity of CD8+ T cells. Tissue microarray analysis in human UC showed an inverse correlation between FGFR3 and PD-L1. Furthermore, NEDD4, an E3 ubiquitin ligase of the NEDD4 family of proteins, was phosphorylated by FGFR3 activation and served as a regulator of PD-L1 ubiquitination. Mechanistically, NEDD4 interacted with PD-L1 and catalyzed Lys48 (K48)-linked polyubiquitination of PD-L1. In mice bearing NEDD4 knockout bladder cancer, CD8+ T-cell infiltration and antitumor activity were significantly inhibited due to PD-L1 upregulation in bladder cancer cells. Furthermore, multiple FGFR3-activated tumor-bearing mouse models suggested that attenuated CD8+ T-cell-mediated antitumor efficacy following FGFR3-targeted therapy could be rescued by a combination with anti-PD-1 immunotherapy, which leads to effective tumor suppression. This study establishes a key molecular link between targeted therapy and immune surveillance and identifies NEDD4 as a crucial E3 ubiquitin ligase that targets PD-L1 for degradation in FGFR3-activated bladder cancer. These findings may potentially be exploited for combination therapies in UC of the bladder and possibly other malignancies with activated FGFR3. SIGNIFICANCE: NEDD4 links two important molecules associated with targeted therapy and immune surveillance, providing mechanistic rationale and preclinical support for immuno-targeted combination therapy for FGFR3-activated bladder cancer.

The nuclear receptor (NR) superfamily is one of the major druggable gene families, representing targets of approximately 13.5% of approved drugs. Certain NRs, such as estrogen receptor and androgen receptor, have been well demonstrated to be functionally involved in cancer and serve as informative biomarkers and therapeutic targets in oncology. However, the spectrum of NR dysregulation across cancers remains to be comprehensively characterized. Through computational integration of genetic, genomic, and pharmacologic profiles, we characterized the expression, recurrent genomic alterations, and cancer dependency of NRs at a large scale across primary tumor specimens and cancer cell lines. Expression levels of NRs were highly cancer-type specific and globally downregulated in tumors compared with corresponding normal tissue. Although the majority of NRs showed copy-number losses in cancer, both recurrent focal gains and losses were identified in select NRs. Recurrent mutations and transcript fusions of NRs were observed in a small portion of cancers, serving as actionable genomic alterations. Analysis of large-scale CRISPR and RNAi screening datasets identified 10 NRs as strongly selective essential genes for cancer cell growth. In a subpopulation of tumor cells, growth dependencies correlated significantly with expression or genomic alterations. Overall, our comprehensive characterization of NRs across cancers may facilitate the identification and prioritization of potential biomarkers and therapeutic targets, as well as the selection of patients for precision cancer treatment. SIGNIFICANCE: Computational analysis of nuclear receptors across multiple cancer types provides a series of biomarkers and therapeutic targets within this protein family.

The growing use of neoadjuvant chemotherapy to treat advanced stage high-grade serous ovarian cancer (HGSOC) creates an opportunity to better understand chemotherapy-induced mutational and gene expression changes. Here we performed a cohort study including 34 patients with advanced stage IIIC or IV HGSOC to assess changes in the tumor genome and transcriptome in women receiving neoadjuvant chemotherapy. RNA sequencing and panel DNA sequencing of 596 cancer-related genes was performed on paired formalin-fixed paraffin-embedded specimens collected before and after chemotherapy, and differentially expressed genes (DEG) and copy-number variations (CNV) in pre- and post-chemotherapy samples were identified. Following tissue and sequencing quality control, the final patient cohort consisted of 32 paired DNA and 20 paired RNA samples. Genomic analysis of paired samples did not reveal any recurrent chemotherapy-induced mutations. Gene expression analyses found that most DEGs were upregulated by chemotherapy, primarily in the chemotherapy-resistant specimens. AP-1 transcription factor family genes (FOS, FOSB, FRA-1) were particularly upregulated in chemotherapy-resistant samples. CNV analysis identified recurrent 11q23.1 amplification, which encompasses SIK2. In vitro, combined treatment with AP-1 or SIK2 inhibitors with carboplatin or paclitaxel demonstrated synergistic effects. These data suggest that AP-1 activity and SIK2 copy-number amplification are induced by chemotherapy and may represent mechanisms by which chemotherapy resistance evolves in HGSOC. AP-1 and SIK2 are druggable targets with available small molecule inhibitors and represent potential targets to circumvent chemotherapy resistance. SIGNIFICANCE: Genomic and transcriptomic analyses identify increased AP-1 activity and SIK2 copy-number amplifications in resistant ovarian cancer following neoadjuvant chemotherapy, uncovering synergistic effects of AP-1 and SIK2 inhibitors with chemotherapy.

Continuous risk of recurrence scores (CRS) based on tumor gene expression are vital prognostic tools for breast cancer. Studies have shown that Black women (BW) have higher CRS than White women (WW). Although systemic injustices contribute substantially to breast cancer disparities, evidence of biological and germline contributions is emerging. In this study, we investigated germline genetic associations with CRS and CRS disparity using approaches modeled after transcriptome-wide association studies (TWAS). In the Carolina Breast Cancer Study, using race-specific predictive models of tumor expression from germline genetics, we performed race-stratified (N = 1,043 WW, 1,083 BW) linear regressions of three CRS (ROR-S: PAM50 subtype score; proliferation score; ROR-P: ROR-S plus proliferation score) on imputed tumor genetically regulated tumor expression (GReX). Bayesian multivariate regression and adaptive shrinkage tested GReX-prioritized genes for associations with tumor PAM50 expression and subtype to elucidate patterns of germline regulation underlying GReX-CRS associations. At FDR-adjusted P < 0.10, 7 and 1 GReX prioritized genes among WW and BW, respectively. Among WW, CRS were positively associated with MCM10, FAM64A, CCNB2, and MMP1 GReX and negatively associated with VAV3, PCSK6, and GNG11 GReX. Among BW, higher MMP1 GReX predicted lower proliferation score and ROR-P. GReX-prioritized gene and PAM50 tumor expression associations highlighted potential mechanisms for GReX-prioritized gene to CRS associations. Among patients with breast cancer, differential germline associations with CRS were found by race, underscoring the need for larger, diverse datasets in molecular studies of breast cancer. These findings also suggest possible germline trans-regulation of PAM50 tumor expression, with potential implications for CRS interpretation in clinical settings. SIGNIFICANCE: This study identifies race-specific genetic associations with breast cancer risk of recurrence scores and suggests mediation of these associations by PAM50 subtype and expression, with implications for clinical interpretation of these scores.

Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. SIGNIFICANCE: Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.

Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

BRCA1/2 mutations account for only a small fraction of homologous recombination (HR) deficiency (HRD) cases. Recently developed genomic HRD (gHRD) tests suffer confounding factors that cause low precision in predicting samples that will respond to PARP inhibitors and DNA damaging agents. Here we present molecular and clinical evidence of transcriptional HRD (tHRD) that is based on aberrant transcript usage (aTU) of minor isoforms. Specifically, increased TU of nonfunctional isoforms of DNA repair genes was prevalent in breast and ovarian cancer with gHRD. Functional assays validated the association of aTU with impaired HR activity. Machine learning-based tHRD detection by the transcript usage (TU) pattern of key genes was superior to directly screening for gHRD or BRCA1/2 mutations in accurately predicting responses of cell lines and patients with cancer to PARP inhibitors and genotoxic drugs. This approach demonstrated the capability of tHRD status to reflect functional HR status, including in a cohort of olaparib-treated ovarian cancer with acquired platinum resistance. Diagnostic tests based on tHRD are expected to broaden the clinical utility of PARP inhibitors. SIGNIFICANCE: A novel but widespread transcriptional mechanism by which homologous recombination deficiency arises independently of BRCA1/2 mutations can be utilized as a companion diagnostic for PARP inhibitors.

Comment in Cancer Res. 2022 Jan 1;82(1):18-20.