Although circular RNAs (circRNA) are known to modulate tumor initiation and progression, their role in hepatocellular carcinoma (HCC) metastasis remains poorly understood. Here, three metastasis-associated circRNAs identified in a previous circRNA-sequencing study were screened and validated in two HCC cohorts. CircRPN2 was downregulated in highly metastatic HCC cell lines and HCC tissues with metastasis. Patients with HCC with lower circRPN2 levels displayed shorter overall survival and higher rates of cumulative recurrence. Mechanistic studies in vitro and in vivo revealed that circRPN2 binds to enolase 1 (ENO1) and accelerates its degradation to promote glycolytic reprogramming through the AKT/mTOR pathway, thereby inhibiting HCC metastasis. CircRPN2 also acted as a competing endogenous RNA for miR-183-5p, which increases forkhead box protein O1 (FOXO1) expression to suppress glucose metabolism and tumor progression. In clinical samples, circRPN2 expression negatively correlated with ENO1 and positively correlated with FOXO1, and expression of circRPN2, either alone or in combination with ENO1 and FOXO1, was a novel indicator of HCC prognosis. These data support a model wherein circRPN2 inhibits HCC aerobic glycolysis and metastasis via acceleration of ENO1 degradation and regulation of the miR-183-5p/FOXO1 axis, suggesting that circRPN2 represents a possible therapeutic target in HCC. SIGNIFICANCE: The circRNA circRPN2 is a potential prognostic biomarker and therapeutic target in hepatocellular carcinoma that suppresses aerobic glycolysis and metastasis.

Bladder cancer is common worldwide, with most patients presenting with nonmuscle invasive disease. Multiple intravesical recurrences lead to reduced quality of life and high costs for patients with this form of bladder cancer. Intravesical chemotherapy aimed at reducing recurrence is the standard-of-care but has significant side effects from nonspecific cytotoxicity to normal urothelium. Importantly, toxicity limits doses that can be administered. Thus, tumor-specific drug targeting could reduce toxicity and enhance effectiveness by allowing higher doses. Here, using cell internalization systematic evolution of ligands by exponential enrichment (SELEX), we identify a novel bladder cancer-specific, chemically modified nucleic acid aptamer that can be preferentially internalized into tumor cells but not normal urothelial cells. The 35-nucleotide B1 aptamer is internalized into bladder cancer cells through clathrin-mediated endocytosis and macropinocytosis. As proof of principle, a B1-guided DNA nanotrain delivery vehicle for epirubicin was constructed as a targeted intravesical chemotherapy. The B1-nanotrain-epirubicin construct exhibited selective cytotoxicity towards bladder cancer cells and outperformed epirubicin in murine orthotopic xenograft models of human bladder cancer. This aptamer-based delivery system makes targeted chemotherapy possible for bladder cancer, providing a compelling rationale for clinical development. SIGNIFICANCE: These findings identify a bladder cancer-specific aptamer that can be used for targeted delivery of chemotherapy, potentially reducing toxicity and enhancing therapeutic efficacy.

Genome-wide association studies (GWAS) have revealed numerous genetic loci associated with colorectal cancer risk, but the mechanisms underlying these loci have not been comprehensively elucidated. In this study, we performed a GWAS meta-analysis with a two-stage replication strategy by combining eight colorectal cancer cohorts encompassing 7,186 cases and 8,512 controls in Chinese populations, accompanied by an evaluation encompassing 29,832 cases and 406,694 controls in European populations. The genetic variant rs505706 A>G, located at chr1q44 in the upstream region of catsper channel auxiliary subunit epsilon (CATSPERE), was associated with colorectal cancer risk and exhibited genome-wide significance (OR, 0.73; 95% confidence interval, 0.67-0.80; P = 9.75 × 10-12). Cell line and animal models were applied to assess the biological function of the genetic risk variant and the corresponding susceptibility gene. Genetically, the G allele of rs505706 resulted in long-range regulatory effects, reducing the binding affinity of POU2F1 for the CATSPERE promoter and thus abolishing the inhibitory effect of POU2F1 on CATSPERE transcription. Phenotypically, CATSPERE upregulation attenuated tumor growth in both colorectal cancer cells and xenograft models. Mechanistically, CATSPERE promoted calcium ion influx and apoptotic pathway activity. In zebrafish models, CATSPERE exerted pleiotropic effects, enhancing the progression of colorectal cancer. Collectively, these findings highlight a colorectal cancer susceptibility locus that acts to remotely modulate the activity of CATSPERE, a gene that mediates multiple functions involved in colorectal tumorigenesis and progression. SIGNIFICANCE: A GWAS meta-analysis identifies a novel susceptibility locus harboring a genetic risk variant that mediates pleiotropic biological effects in colorectal tumorigenesis and progression.

Comment on Cancer Res. 2022 Mar 15;82(6):1013-1024.

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and Rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2-M cell-cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited break-induced replication and homologous recombination repair. In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM pathway or that harbor putative drivers of replication stress such as CCNE1 amplification. This translated to in vivo antitumor activity, with tumor control requiring continuous dosing and free plasma exposures, which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimization of dose and schedules in vivo and showed superior antitumor activity at lower doses compared with that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twice daily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3 to5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent. SIGNIFICANCE: This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

Comment in Cancer Res. 2022 Mar 15;82(6):969-971.

Comment on Cancer Res. 2022 Mar 15;82(6):1025-1037.

Because of profound effects observed in carcinogenesis, prostaglandins (PG), prostaglandin-endoperoxide synthases, and PG receptors are implicated in cancer development and progression. Understanding the molecular mechanisms of PG actions has potential clinical relevance for cancer prevention and therapy. This review focuses on the current status of PG signaling pathways in modulating cancer progression and aims to provide insights into the mechanistic actions of PGs and their receptors in influencing tumor progression. We also examine several small molecules identified as having anticancer activity that target prostaglandin receptors. The literature suggests that targeting PG pathways could provide opportunities for cancer prevention and therapy.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy. Although more than half of patients with DLBCL achieve long-term remission, the majority of remaining patients succumb to the disease. As abnormal iron homeostasis is implicated in carcinogenesis and the progression of many tumors, we searched for alterations in iron metabolism in DLBCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of large cohorts of patients with DLBCL established the iron score (IS), a gene expression-based risk score enabling identification of patients with DLBCL with a poor outcome who might benefit from a suitable targeted therapy. In a panel of 16 DLBCL cell lines, ironomycin, a promising lysosomal iron-targeting small molecule, inhibited DLBCL cell proliferation at nanomolar concentrations compared with typical iron chelators. Ironomycin also induced significant cell growth inhibition, ferroptosis, and autophagy. Ironomycin treatment resulted in accumulation of DNA double-strand breaks, delayed progression of replication forks, and increased RPA2 phosphorylation, a marker of replication stress. Ironomycin significantly reduced the median number of viable primary DLBCL cells of patients without major toxicity for nontumor cells from the microenvironment and presented low toxicity in hematopoietic progenitors compared with conventional treatments. Significant synergistic effects were also observed by combining ironomycin with doxorubicin, BH3 mimetics, BTK inhibitors, or Syk inhibitors. Altogether, these data demonstrate that a subgroup of high-risk patients with DLBCL can be identified with the IS that can potentially benefit from targeting iron homeostasis. SIGNIFICANCE: Iron homeostasis represents a potential therapeutic target for high-risk patients with DLBCL that can be targeted with ironomycin to induce cell death and to sensitize tumor cells to conventional treatments.

The outcomes of adolescents/young adults with osteosarcoma have not improved in decades. The chaotic karyotype of this rare tumor has precluded the identification of prognostic biomarkers and patient stratification. We reasoned that transcriptomic studies should overcome this genetic complexity. RNA sequencing (RNA-seq) of 79 osteosarcoma diagnostic biopsies identified stable independent components that recapitulate the tumor and microenvironment cell composition. Unsupervised classification of the independent components stratified this cohort into favorable (G1) and unfavorable (G2) prognostic tumors in terms of overall survival. Multivariate survival analysis ranked this stratification as the most influential variable. Functional characterization associated G1 tumors with innate immunity and G2 tumors with angiogenic, osteoclastic, and adipogenic activities as well as PPARγ pathway upregulation. A focused gene signature that predicted G1/G2 tumors from RNA-seq data was developed and validated within an independent cohort of 82 osteosarcomas. This signature was further validated with a custom NanoString panel in 96 additional osteosarcomas. This study thus proposes new biomarkers to detect high-risk patients and new therapeutic options for osteosarcoma. SIGNIFICANCE: These findings indicate that the osteosarcoma microenvironment composition is a major feature to identify hard-to-treat patient tumors at diagnosis and define the biological pathways and potential actionable targets associated with these tumors.

Comment in Cancer Res. 2022 Mar 15;82(6):972-973.

In the mid 1990's, a convergence of discoveries in dendritic cell (DC) biology and tumor antigen identification led investigators to study DCs as adjuvants for cancer vaccines. On the twentieth anniversary of a seminal clinical study by Jacques Banchereau and colleagues, we revisit the key events that prompted the initial wave of DC vaccine clinical studies and lessons learned that, in our opinion, helped forge the path for the field that we now call immuno-oncology. It is essential to recall that prior to the discovery of immune checkpoint therapy and chimeric antigen receptor (CAR) T-cell therapy, skepticism prevailed regarding the potential therapeutic benefit of immunotherapies. In hindsight, we can now appreciate how the early DC cancer vaccine trials helped investigators sustain their attention on adaptive immunity specific for malignant cells. These vaccines demonstrated clear evidence for induction of antigen-specific T cells and were well tolerated despite low rates of objective clinical response. In the context of the current era some 20 years later, harnessing DC vaccines has been shown to increase the breadth and diversity of tumor-specific T cells, and by trafficking to sites of metastases promote an inflamed tumor microenvironment. See related article by Banchereau and colleagues, Cancer Res 2001; 61:6451-8.

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors. SIGNIFICANCE: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

Preventing development of childhood B-cell acute lymphoblastic leukemia (B-ALL), a disease with devastating effects, is a longstanding and unsolved challenge. Heterozygous germline alterations in the PAX5 gene can lead to B-ALL upon accumulation of secondary mutations affecting the JAK/STAT signaling pathway. Preclinical studies have shown that this malignant transformation occurs only under immune stress such as exposure to infectious pathogens. Here we show in Pax5+/- mice that transient, early-life administration of clinically relevant doses of ruxolitinib, a JAK1/2 inhibitor, significantly mitigates the risk of B-ALL following exposure to infection; 1 of 29 animals treated with ruxolitinib developed B-ALL versus 8 of 34 untreated mice. Ruxolitinib treatment preferentially targeted Pax5+/- versus wild-type B-cell progenitors and exerted unique effects on the Pax5+/- B-cell progenitor transcriptional program. These findings provide the first in vivo evidence for a potential strategy to prevent B-ALL development. SIGNIFICANCE: JAK/STAT inhibition suppresses tumorigenesis in a B-ALL-susceptible mouse model, presenting a novel approach to prevent B-ALL onset.

Kinase fusions have been identified in a growing subset of sarcomas, but a lack of preclinical models has impeded their functional analysis as therapeutic targets in the sarcoma setting. In this study, we generated models of sarcomas bearing kinase fusions and assessed their response to molecularly targeted therapy. Immortalized, untransformed human mesenchymal stem cells (HMSC), a putative cell of origin of sarcomas, were modified using CRISPR-Cas9 to harbor a RET chromosomal translocation (HMSC-RET). In parallel, patient-derived models of RET- and NTRK-rearranged sarcomas were generated. Expression of a RET fusion activated common proliferation and survival pathways and transformed HMSC cells. The HMSC-RET models displayed similar behavior and response to therapy as the patient-derived counterparts in vitro and in vivo. Capicua (CIC)-mediated suppression of negative MAPK pathway regulators was identified as a potential mechanism by which these sarcomas compensate for RET or NTRK inhibition. This CIC-mediated feedback reactivation was blocked by coinhibition of the MAPK pathway and RET or NTRK in the respective models. Importantly, the combination of RET and ERK inhibitors was more effective than single agents at blocking tumor growth in vivo. This work offers new tools and insights to improve targeted therapy approaches in kinase-addicted sarcomas and supports upfront combination therapy to prolong responses. SIGNIFICANCE: Novel models of kinase-rearranged sarcomas show that MAPK pathway feedback activation dampens responses to tyrosine kinase inhibitors, revealing the potential of combinatorial therapies to combat these tumors.

Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine-rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody-drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases. Significance: This study identifies that LRRC15 activates β1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody-drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.

Nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) are two major EBV-associated epithelial malignancies, both of which are characterized by the infiltration of a large number of lymphocytes, including natural killer (NK) cells. Although NK cells can prevent the development of EBV-associated epithelial malignancies, EBV-infected tumor cells often develop resistance to surveillance by NK cells. Elucidating the interactions between NK cells and EBV-infected tumor cells will facilitate the development of more effective NK-mediated therapies for treating EBV-associated malignancies. Here we investigated the cytotoxic function of NK cells in EBV-associated epithelial malignancies and discovered that EBV infection-induced upregulation of F3 expression correlates with NK-cell dysfunction in NPC and EBVaGC. The subsequent inhibitory effect of F3-mediated platelet aggregation on NK-cell function was verified in vitro and in vivo. Mechanistically, EBV latent membrane protein 2A (LMP2A) mediated upregulation of F3 through the PI3K/AKT signaling pathway. In an NPC xenograft mouse model, inhibition of F3 restored the antitumor function of NK cells and showed therapeutic efficacy when administered with NK-cell transfer. On the basis of these findings, EBV infection induces F3-mediated platelet aggregation that inhibits the antitumor function of NK cells, providing a rationale for developing and combining NK-cell-based therapies with F3 inhibitors to treat EBV-associated epithelial malignancies. SIGNIFICANCE: This study reveals a mechanism by which EBV-associated epithelial malignancies escape NK-cell-mediated immune surveillance, providing a new target for improving NK-cell immunotherapy.

Docetaxel-based chemotherapy is a standard-of-care treatment for metastatic prostate cancer, and chemoresistance remains a major challenge in clinical practice. Recent studies have demonstrated that circular RNAs (circRNA) play critical roles in the development and progression of prostate cancer. However, the biological roles and potential functions of circRNAs in mediating docetaxel-resistant prostate cancer have yet to be well elucidated. In this study, we analyzed the expression profiles of circRNAs in docetaxel-resistant and -sensitive prostate cancer cells through RNA sequencing and found that expression of circARHGAP29 was significantly upregulated in docetaxel-resistant cell lines and clinical samples. Ectopic expression of circARHGAP29 triggered docetaxel resistance and aerobic glycolysis in prostate cancer cells, which was reduced by silencing circARHGAP29. Moreover, eukaryotic initiation factor 4A3, which bound the back-spliced junction site and the downstream flanking sequence of circARHGAP29, induced cyclization and cytoplasmic export of circARHGAP29. circARHGAP29 increased the stability of lactate dehydrogenase A (LDHA) mRNA by strengthening its interaction with insulin-like growth factor 2 mRNA-binding protein 2, leading to enhanced glycolytic metabolism. In addition, circARHGAP29 interacted with and stabilized c-Myc mRNA and protein, which further increased LDHA expression by facilitating its transcription. These findings reveal the crucial function of circARHGAP29 in prostate cancer glycolysis by increasing and stabilizing LDHA mRNA, providing a promising therapeutic target in docetaxel-resistant prostate cancer. SIGNIFICANCE: Upregulation of a novel circRNA, circARHGAP29, promotes docetaxel resistance and glycolytic metabolism, suggesting it could be a prognostic biomarker and therapeutic target in chemoresistant prostate cancer.

Breast cancer is the most common malignancy in women worldwide. However, the mechanisms underlying breast cancer energy metabolism and progression remain obscure. Cancer cells rapidly adapt to microenvironments with fluctuating nutrient levels. Here, we characterized a long noncoding RNA (lncRNA), HOXC-AS3, which is activated upon glucose deprivation to trigger a nutrient-stress response and a switch in glucose metabolism. Upregulation of HOXC-AS3 in breast cancer was identified by in published microarray and RNA-sequencing datasets, and then confirmed by qRT-PCR in fresh breast cancer tissues. Glucose deprivation induced HOXC-AS3 overexpression in a dose- and time-dependent manner in breast cancer cells. Gain- and loss-of-function experiments in vitro and in vivo showed that HOXC-AS3 triggers energy metabolism reprogramming. ChIRP-mass spectrometry and unique molecular identifier RNA immunoprecipitation and high-throughput sequencing (UMI RIP-seq) identified binding motifs of HOXC-AS3 with SIRT6. HOXC-AS3 selectively antagonized SIRT6-mediated H3K9ac deacetylation of glycolysis-related genes. Moreover, HOXC-AS3 binding to SIRT6 prevented contact inhibition of HIF1α, leading to reprogramming of metabolic pathways. In addition, HOXC-AS3, SP1, and miR-1224-5p formed a positive feedback loop to maintain cancer-promoting signals. Furthermore, administration of anti-HOXC-AS3-motif-RNAs effectively blocked the function of HOXC-AS3, ultimately suppressing breast cancer progression. These results reveal a critical role for HOXC-AS3 in regulating the metabolic reprogramming of breast cancer cells under metabolic stress. Use of an anti-HOXC-AS3-motif RNA mixture may be a promising strategy to suppress breast cancer progression. SIGNIFICANCE: HOXC-AS3 is a low glucose-activated long noncoding RNA that triggers metabolic reprogramming in breast cancer cells to adapt to nutrient stress, identifying HOXC-AS3 as a potential therapeutic target for breast cancer treatment.

Mutations in SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically. SIGNIFICANCE: The cancer-associated SF3B1K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.

The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in regulation of the Warburg effect, which is characterized by the preference for aerobic glycolysis over oxidative phosphorylation for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Antisense oligonucleotides (ASO) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform have been shown to induce apoptosis in cultured glioblastoma cells when delivered by lipofection. Here, we explore the potential of ASO-based PKM splice switching as a targeted therapy for liver cancer. A more potent lead constrained-ethyl (cEt)/DNA ASO induced PKM splice switching and inhibited the growth of cultured hepatocellular carcinoma (HCC) cells. This PKM isoform switch increased pyruvate-kinase activity and altered glucose metabolism. In an orthotopic HCC xenograft mouse model, the lead ASO and a second ASO targeting a nonoverlapping site inhibited tumor growth. Finally, in a genetic HCC mouse model, a surrogate mouse-specific ASO induced Pkm splice switching and inhibited tumorigenesis, without observable toxicity. These results lay the groundwork for a potential ASO-based splicing therapy for HCC. SIGNIFICANCE: Antisense oligonucleotides are used to induce a change in PKM isoform usage in hepatocellular carcinoma, reversing the Warburg effect and inhibiting tumorigenesis.

Dormant cancer cells that survive anticancer therapy can lead to cancer recurrence and disseminated metastases that prove fatal in most cases. Recently, specific dormant polyploid giant cancer cells (PGCC) have drawn our attention because of their association with the clinical risk of nasopharyngeal carcinoma (NPC) recurrence, as demonstrated by previous clinical data. In this study, we report the biological properties of PGCC, including mitochondrial alterations, and reveal that autophagy is a critical mechanism of PGCC induction. Moreover, pharmacologic or genetic inhibition of autophagy greatly impaired PGCC formation, significantly suppressing metastasis and improving survival in a mouse model. Mechanistically, chemotherapeutic drugs partly damaged mitochondria, which then produced low ATP levels and activated autophagy via the AMPK-mTOR pathway to promote PGCC formation. Analysis of the transcriptional and epigenetic landscape of PGCC revealed overexpression of RIPK1, and the scaffolding function of RIPK1 was required for AMPK-mTOR pathway-induced PGCC survival. High numbers of PGCCs correlated with shorter recurrence time and worse survival outcomes in patients with NPC. Collectively, these findings suggest a therapeutic approach of targeting dormant PGCCs in cancer. SIGNIFICANCE: Pretreatment with an autophagy inhibitor before chemotherapy could prevent formation of therapy-induced dormant polyploid giant cancer cells, thereby reducing recurrence and metastasis of nasopharyngeal carcinoma.

Primary myelofibrosis (PMF) is one of three myeloproliferative neoplasms (MPN) that are morphologically and molecularly inter-related, the other two being polycythemia vera (PV) and essential thrombocythemia (ET). MPNs are characterized by JAK-STAT-activating JAK2, CALR, or MPL mutations that give rise to stem cell-derived clonal myeloproliferation, which is prone to leukemic and, in case of PV and ET, fibrotic transformation. Abnormal megakaryocyte proliferation is accompanied by bone marrow fibrosis and characterizes PMF, while the clinical phenotype is pathogenetically linked to ineffective hematopoiesis and aberrant cytokine expression. Among MPN-associated driver mutations, type 1-like CALR mutation has been associated with favorable prognosis in PMF, while ASXL1, SRSF2, U2AF1-Q157, EZH2, CBL, and K/NRAS mutations have been shown to be prognostically detrimental. Such information has enabled development of exclusively genetic (GIPSS) and clinically integrated (MIPSSv2) prognostic models that facilitate individualized treatment decisions. Allogeneic stem cell transplantation remains the only treatment modality in MF with the potential to prolong survival, whereas drug therapy, including JAK2 inhibitors, is directed mostly at the inflammatory component of the disease and is therefore palliative in nature. Similarly, disease-modifying activity remains elusive for currently available investigational drugs, while their additional value in symptom management awaits controlled confirmation. There is a need for genetic characterization of clinical observations followed by in vitro and in vivo preclinical studies that will hopefully identify therapies that target the malignant clone in MF to improve patient outcomes.

ARID1A is a key mammalian SWI/SNF complex subunit that is mutated in 5% to 11% of lung cancers. Although recent studies have elucidated the mechanism underlying dysregulation of the switch/sucrose non-fermentable (SWI/SNF) complexes in cancers, the significance of ARID1A loss and its implications in lung cancers remain poorly defined. This study investigates how ARID1A loss affects initiation and progression of lung cancer. In genetically engineered mouse models bearing mutant Kras and a deficient Trp53 allele (KP), ARID1A loss (KPA) promoted lung tumorigenesis. Analysis of the transcriptome profiles of KP and KPA tumors suggested enhanced glycolysis following ARID1A loss, and expression of the glycolytic regulators Pgam1, pyruvate kinase M (Pkm), and Pgk1 was significantly increased in ARID1A-deficient lung tumors. Furthermore, ARID1A loss increased chromatin accessibility and enhanced hypoxia-inducible factor-1α (HIF1α) binding to the promoter regions of Pgam1, Pkm, and Pgk1. Loss of ARID1A in lung adenocarcinoma also resulted in loss of histone deacetylase 1 (HDAC1) recruitment, increasing acetylation of histone-4 lysine at the promoters of Pgam1, Pkm, and Pgk1, and subsequently enhancing BRD4-driven transcription of these genes. Metabolic analyses confirmed that glycolysis is enhanced in ARID1A-deficient tumors, and genetic or pharmacologic inhibition of glycolysis inhibited lung tumorigenesis in KPA mice. Treatment with the small molecule bromodomain and extraterminal protein (BET) inhibitor JQ1 compromised both initiation and progression of ARID1A-deficient lung adenocarcinoma. ARID1A negatively correlated with glycolysis-related genes in human lung adenocarcinoma. Overall, ARID1A loss leads to metabolic reprogramming that supports tumorigenesis but also confers a therapeutic vulnerability that could be harnessed to improve the treatment of ARID1A-deficient lung cancer. SIGNIFICANCE: This study links ARID1A loss with enhanced glycolysis in lung cancer and demonstrates the preclinical efficacy of BET inhibitor therapy as a strategy to combat tumor growth.

DEAD-box RNA helicases belong to a large group of RNA-processing factors and play vital roles unwinding RNA helices and in ribosomal RNA biogenesis. Emerging evidence indicates that RNA helicases are associated with genome stability, yet the mechanisms behind this association remain poorly understood. In this study, we performed a comprehensive analysis of RNA helicases using multiplatform proteogenomic databases. More than 50% (28/49) of detected RNA helicases were highly expressed in multiple tumor tissues, and more than 60% (17/28) of tumor-associated members were directly involved in DNA damage repair (DDR). Analysis of repair dynamics revealed that these RNA helicases are engaged in an extensively broad range of DDR pathways. Among these factors is DDX21, which was prominently upregulated in colorectal cancer. The high expression of DDX21 gave rise to frequent chromosome exchange and increased genome fragmentation. Mechanistically, aberrantly high expression of DDX21 triggered inappropriate repair processes by delaying homologous recombination repair and increasing replication stress, leading to genome instability and tumorigenesis. Treatment with distinct chemotherapeutic drugs caused higher lethality to cancer cells with genome fragility induced by DDX21, providing a perspective for treatment of tumors with high DDX21 expression. This study revealed the role of RNA helicases in DNA damage and their associations with cancer, which could expand therapeutic strategies and improve precision treatments for cancer patients with high expression of RNA helicases. SIGNIFICANCE: The involvement of the majority of tumor-associated RNA helicases in the DNA damage repair process suggests a new mechanism of tumorigenesis and offers potential alternative therapeutic strategies for cancer.

Antibody-peptide epitope conjugates (APEC) are a new class of modified antibody-drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs. SIGNIFICANCE: This study develops a high-throughput preclinical platform to identify patient-specific antibody-peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

Tumor dormancy is a stage in which residual cancer cells remain inactive, but regrowth of dormant cancer cells contributes to recurrence. The complex ecosystem in cancer that promotes cell survival and the factors that eventually overcome growth constraints and result in proliferation remain to be fully elucidated. Doing so may provide new insights and help identify novel strategies to prolong cancer dormancy and prevent disease recurrence. To dissect the molecular pathways and the microenvironments involved in regulation of dormancy, we utilized a novel immunocompetent transgenic model to study minimal residual disease and relapse. This model revealed a significant reorganization of cancer cell structures, stroma, and immune cells, with cancer cells showing dormant cell signatures. Single-cell RNA sequencing uncovered remodeling of myeloid and lymphoid compartments. In addition, the Jagged-1/Notch signaling pathway was shown to regulate many aspects of tumorigenesis, including stem cell development, epithelial-to-mesenchymal transition, and immune cell homeostasis during minimal residual disease. Treatment with an anti-Jagged-1 antibody inhibited the Jagged-1/Notch signaling pathway in tumor cells and the microenvironment, delaying tumor recurrence. These findings uncover a cascade of regulatory changes in the microenvironment during dormancy and identify a therapeutic strategy to undercut these changes. SIGNIFICANCE: Single-cell RNA-sequencing analysis reveals dormancy-associated changes in immune and stromal cells and demonstrates a rationale to pursue Jagged-1/Notch pathway inhibition as a viable therapeutic strategy to reduce disease recurrence.

Enhancer RNAs (eRNA) regulate gene expression and play critical roles in cancer. Using large-scale omics data from The Cancer Genome Atlas (TCGA), we systematically investigated the impact of genetic variants on eRNA expression and identified approximately 1 million eRNA quantitative trait loci (eRNA-QTL) as cis- and trans-acting. Over 16,000 eRNA-QTLs were associated with patient overall survival. Assessing the impact of eRNAs on more than 1,000 imputed anticancer drug responses across approximately 10,000 patients with cancer revealed more than 7 million significant associations. Furthermore, approximately 240,000 significant associations were identified between eRNA expression and immune cell abundance deconvoluted by TIMER, CIBERSORT, ImmuCellAI, and ImmuneCellGSVA. Finally, a user-friendly data portal was generated: Genetic, Pharmacogenomic, and Immune Landscapes of eRNAs (GPIeR; https://hanlab.tamhsc.edu/GPIeR/). GPIeR is a large-scale multidimensional data portal that can be used to explore eRNA-associated genetic variants, drug responses, and immune infiltration, with the purpose of facilitating functional and clinical investigations of eRNAs in cancer. SIGNIFICANCE: This study provides insights into the impact of the enhancer RNA (eRNA) landscape on patient outcome, the immune microenvironment, and drug response across cancers and develops a multidimensional data portal to facilitate eRNA research.

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to "BRCAness" in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC. SIGNIFICANCE: In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.

Comment on Cancer Res. 2022 Mar 1;82(5):859-871.

Comment on Cancer Res. 2022 Mar 1;82(5):929-942.

Over the past two decades, there have been advances in surgical technologies and chemoradiation strategies for glioblastoma, yet durable remissions are rarely seen. As the biological challenges and genetic basis of glioblastoma have become more understood, new therapeutic strategies may lead to more durable clinical responses and long-term remissions. We believe specialized academic centers that form meaningful corporate partnerships to complement basic science infrastructure and use adaptive clinical trial designs will achieve more rapid translation of innovative approaches to glioblastoma. Here we outline the core biological challenges to be overcome in the management of glioblastoma.

Tetraploidy is an aneuploidy-permissive condition that can fuel tumorgenesis. The tip-over hypothesis of cytotoxic therapy sensitivity proposes that therapy is effective if it pushes a cell's aneuploidy above a viable tipping point. But elevated aneuploidy alone may not account for this tipping point. Tissue microenvironments that lack sufficient resources to support tetraploid cells can explain the fitness cost of aneuploidy. Raw materials needed to generate deoxynucleotides, the building blocks of DNA, are candidate rate-limiting factors for the evolution of high-ploidy cancer cells. Understanding the resource cost of high ploidy is key to uncover its therapeutic vulnerabilities across tissue sites with versatile energy supplies.

Comment in Cancer Res. 2022 Mar 1;82(5):766-768.

Comment in Cancer Res. 2022 Mar 1;82(5):764-765.