Somatic mutations of MLL4/COMPASS induce cytoplasmic localization providing molecular insight into cancer prognosis and treatment.

Cancer genome sequencing consortiums have recently catalogued an abundance of somatic mutations, across a wide range of human cancers, in the chromatin-modifying enzymes that regulate gene expression. Defining the molecular mechanisms underlying the potentially oncogenic functions of these epigenetic mutations could serve as the basis for precision medicine approaches to cancer therapy. MLL4 encoded by the KMT2D gene highly mutated in a large number of human cancers, is a key histone lysine monomethyltransferase within the Complex of Proteins Associated with Set1 (COMPASS) family that regulates gene expression through enhancer function, potentially functioning as a tumor suppressor. We report that the KMT2D mutations which cause MLL4 protein truncation also alter MLL4's subcellular localization, resulting in loss-of-function in the nucleus and gain-of-function in the cytoplasm. We demonstrate that isogenic correction of KMT2D truncation mutation rescues the aberrant localization phenotype and restores multiple regulatory functions of MLL4, including COMPASS integrity/stabilization, histone H3K4 mono-methylation, enhancer activation, and therefore transcriptional regulation. Moreover, isogenic correction diminishes the sensitivity of KMT2D-mutated cancer cells to targeted metabolic inhibition. Using immunohistochemistry, we identified that cytoplasmic MLL4 is unique to the tissue of bladder cancer patients with KMT2D truncation mutations. Using a preclinical carcinogen model of bladder cancer in mouse, we demonstrate that truncated cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D-mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome.

DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells.

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Spatial comparison of molecular features associated with resistance to pembrolizumab in BCG unresponsive bladder cancer.

Intravenous immune checkpoint inhibition achieves a 40% 3-month response in BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ. Yet, only half of the early responders will continue to be disease-free by 12 months, and resistance mechanisms are poorly defined. We performed spatial profiling of BCG-unresponsive tumors from patients responsive or resistant to intravenous pembrolizumab treatment, analyzing samples both before initiating and 3 months post-intravenous pembrolizumab treatment. We analyzed 119 regions of interest, which included 59 pairs of epithelial and adjacent stromal segments across five patients: two responders and three non-responders. We demonstrate that BCG unresponsive tumors with an inflamed PanCK+ tumor area and an infiltrated stromal segment respond better to intravenous pembrolizumab. Furthermore, using segment-specific gene signatures generated from a cohort of BCG unresponsive NMIBC treated with intravesical BCG+pembrolizumab, we find that non-inflamed, immune-cold tumors that do not respond to intravenous pembrolizumab exhibit a favorable outcome to the combined application of BCG and pembrolizumab. For the first time, we have identified molecular features of tumors associated with response and resistance to intravenous pembrolizumab in BCG unresponsive NMIBCs. Further research with more patients and alternative checkpoint inhibitors is essential to validate our findings. We anticipate that using a transcriptomics signature like the one described here can help identify tumors with a higher possibility of responding to intravenous pembrolizumab.

A Spatial Comparison of Molecular Features Associated with Resistance to Pembrolizumab in BCG Unresponsive Bladder Cancer.

Background: Intravenous immune checkpoint inhibition achieves a 40% three-month response in BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS). Yet only half of early responders will continue to be disease free by 12 months, and resistance mechanisms are poorly defined. Objective: We assessed the molecular features associated with response to immunotherapy in BCG unresponsive non-muscle invasive bladder cancers treated with pembrolizumab. Design Setting and Participants: We performed digital spatial profiling (DSP) of BCG unresponsive NMIBC tumors before and after IV pembrolizumab therapy. Intervention: Pembrolizumab was administered intravenously in patients with NMIBC at the time of recurrence after BCG therapy. Biopsies were obtained before starting IV pembrolizumab and three months post-treatment. Outcomes and Statistical Analysis: Spatial gene expression profiling of the tumor niche pre- and post IV pembrolizumab. Results and Limitations: We evaluated 119 regions of interest (ROIs) from five patients, which included 60 epithelial (PanCK+) and 59 stromal segments (PanCK-). ROIs from responders had distinct expression signatures from non-responders for both the tumor and TME. Responders were more likely to have a dynamic change in expression after pembrolizumab than non-responders. A major limitation of this study was the number of patients evaluated. Conclusion: For the first time, we have identified distinct expression signatures associated with response and resistance to IV pembrolizumab in NMIBCs. Further research with more patients and alternative checkpoint inhibitors is essential to validate our findings. Patient Summary: We identify the molecular features of tumors associated with response to pembrolizumab for patients with BCG unresponsive NMIBCs.

Expression-based subtypes define pathologic response to neoadjuvant immune-checkpoint inhibitors in muscle-invasive bladder cancer.

Checkpoint immunotherapy (CPI) has increased survival for some patients with advanced-stage bladder cancer (BCa). However, most patients do not respond. Here, we characterized the tumor and immune microenvironment in pre- and post-treatment tumors from the PURE01 neoadjuvant pembrolizumab immunotherapy trial, using a consolidative approach that combined transcriptional and genetic profiling with digital spatial profiling. We identify five distinctive genetic and transcriptomic programs and validate these in an independent neoadjuvant CPI trial to identify the features of response or resistance to CPI. By modeling the regulatory network, we identify the histone demethylase KDM5B as a repressor of tumor immune signaling pathways in one resistant subtype (S1, Luminal-excluded) and demonstrate that inhibition of KDM5B enhances immunogenicity in FGFR3-mutated BCa cells. Our study identifies signatures associated with response to CPI that can be used to molecularly stratify patients and suggests therapeutic alternatives for subtypes with poor response to neoadjuvant immunotherapy.

Role of Chromatin Modifying Complexes and Therapeutic Opportunities in Bladder Cancer.

BACKGROUND: Chromatin modifying enzymes, mainly through post translational modifications, regulate chromatin architecture and by extension the underlying transcriptional kinetics in normal and malignant cells. Muscle invasive bladder cancer (MIBC) has a high frequency of alterations in chromatin modifiers, with 76% of tumors exhibiting mutation in at least one chromatin modifying enzyme [1]. Additionally, clonal expansion of cells with inactivating mutations in chromatin modifiers has been identified in the normal urothelium, pointing to a currently unknown role of these proteins in normal bladder homeostasis. OBJECTIVE: To review current knowledge of chromatin modifications and enzymes regulating these processes in Bladder cancer (BCa). METHODS: By reviewing current literature, we summarize our present knowledge of external stimuli that trigger loss of equilibrium in the chromatin accessibility landscape and emerging therapeutic interventions for targeting these processes. RESULTS: Genetic lesions in BCa lead to altered function of chromatin modifying enzymes, resulting in coordinated dysregulation of epigenetic processes with disease progression. CONCLUSION: Mutations in chromatin modifying enzymes are wide-spread in BCa and several promising therapeutic targets for modulating activity of these genes are currently in clinical trials. Further research into understanding how the epigenetic landscape evolves as the disease progresses, could help identify patients who might benefit the most from these targeted therapies.

First-in-human Intravesical Delivery of Pembrolizumab Identifies Immune Activation in Bladder Cancer Unresponsive to Bacillus Calmette-Guérin.

BACKGROUND: Intravenous immune checkpoint inhibition is an effective anticancer strategy for bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC) but may be associated with greater systemic toxicity compared with localized therapies. OBJECTIVE: We assessed the safety and antitumor activity of intravesical pembrolizumab combined with BCG. DESIGN, SETTING, AND PARTICIPANTS: A 3 + 3 phase 1 trial of pembrolizumab + BCG was conducted in patients with BCG-unresponsive NMIBC (NCT02808143). INTERVENTION: Pembrolizumab was given intravesically (1-5 mg/kg for 2 h) beginning 2 weeks prior to BCG induction until recurrence. Urine profiling during treatment and spatial transcriptomic profiling of pre- and post-treatment tumors were conducted to identify biomarkers that correlated with response. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Safety and tolerability of immune checkpoint inhibition were assessed, and Kaplan-Meier survival analysis was performed. RESULTS AND LIMITATIONS: Nine patients completed therapy. Median follow-up was 35 months for five patients still alive at the end of the trial. The trial was closed due to the COVID-19 pandemic. Grade 1-2 urinary symptoms were common. The maximum tolerated dose was not reached; however, one dose-limiting toxicity was reported (grade 2 diarrhea) in the only patient who reached 52 weeks without recurrence. One death occurred from myasthenia gravis that was deemed potentially related to treatment. The 6-mo and 1-yr recurrence-free rates were 67% (95% confidence interval [CI]: 42-100%) and 22% (95% CI: 6.5-75%), respectively. Pembrolizumab was detected in the urine and not in blood. CD4+ T cells were significantly increased in the urine after treatment, and a transcriptomic analysis identified decreased expression of T-cell exhaustion markers in late recurrences. CONCLUSIONS: We demonstrate that intravesical pembrolizumab is safe, feasible, and capable of eliciting strong immune responses in a clinical setting and should be investigated further. PATIENT SUMMARY: Direct application of pembrolizumab to the bladder is a promising alternative for non-muscle-invasive bladder cancer unresponsive to Bacillus Calmette-Guérin and should be investigated further.

Immune activation is essential for the antitumor activity of EZH2 inhibition in urothelial carcinoma.

The long-term survival of patients with advanced urothelial carcinoma (UCa) is limited because of innate resistance to treatment. We identified elevated expression of the histone methyltransferase EZH2 as a hallmark of aggressive UCa and hypothesized that EZH2 inhibition, via a small-molecule catalytic inhibitor, might have antitumor effects in UCa. Here, in a carcinogen-induced mouse bladder cancer model, a reduction in tumor progression and an increase in immune infiltration upon EZH2 inhibition were observed. Treatment of mice with EZH2i causes an increase in MHC class II expression in the urothelium and can activate infiltrating T cells. Unexpectedly, we found that the lack of an intact adaptive immune system completely abolishes the antitumor effects induced by EZH2 catalytic inhibition. These findings show that immune evasion is the only important determinant for the efficacy of EZH2 catalytic inhibition treatment in a UCa model.

Biodegradable nanoparticles induce cGAS/STING-dependent reprogramming of myeloid cells to promote tumor immunotherapy.

Cancer treatment utilizing infusion therapies to enhance the patient's own immune response against the tumor have shown significant functionality in a small subpopulation of patients. Additionally, advances have been made in the utilization of nanotechnology for the treatment of disease. We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

Multifaceted Impact of MicroRNA 493-5p on Genome-Stabilizing Pathways Induces Platinum and PARP Inhibitor Resistance in BRCA2-Mutated Carcinomas.

BRCA1/2-mutated ovarian cancers (OCs) are defective in homologous recombination repair (HRR) of double-strand breaks (DSBs) and thereby sensitive to platinum and PARP inhibitors (PARPis). Multiple PARPis have recently received US Food and Drug Administration (FDA) approval for treatment of OCs, and resistance to PARPis is a major clinical problem. Utilizing primary and recurrent BRCA1/2-mutated carcinomas from OC patients, patient-derived lines, and an in vivo BRCA2-mutated mouse model, we identified a microRNA, miR-493-5p, that induced platinum/PARPi resistance exclusively in BRCA2-mutated carcinomas. However, in contrast to the most prevalent resistance mechanisms in BRCA mutant carcinomas, miR-493-5p did not restore HRR. Expression of miR-493-5p in BRCA2-mutated/depleted cells reduced levels of nucleases and other factors involved in maintaining genomic stability. This resulted in relatively stable replication forks, diminished single-strand annealing of DSBs, and increased R-loop formation. We conclude that impact of miR-493-5p on multiple pathways pertinent to genome stability cumulatively causes PARPi/platinum resistance in BRCA2 mutant carcinomas.

Targeting MYC dependency in ovarian cancer through inhibition of CDK7 and CDK12/13.

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.

Evolutionarily conserved serum microRNAs predict radiation-induced fatality in nonhuman primates.

Effective planning for the medical response to a radiological or nuclear accident is complex. Because of limited resources for medical countermeasures, the key would be to accurately triage and identify victims most likely to benefit from treatment. We used a mouse model system to provide evidence that serum microRNAs (miRNAs) may effectively predict the impact of radiation on the long-term viability of animals. We had previously used nonhuman primates (NHPs) to demonstrate that this concept is conserved and serum miRNA signatures have the potential to serve as prediction biomarkers for radiation-induced fatality in a human population. We identified a signature of seven miRNAs that are altered by irradiation in both mice and NHPs. Genomic analysis of these conserved miRNAs revealed that there is a combination of seven transcription factors that are predicted to regulate these miRNAs in human, mice, and NHPs. Moreover, a combination of three miRNAs (miR-133b, miR-215, and miR-375) can identify, with nearly complete accuracy, NHPs exposed to radiation versus unexposed NHPs. Consistent with historical data, female macaques appeared to be more sensitive to radiation, but the difference was not significant. Sex-based stratification allowed us to identify an interaction between gender and miR-16-2 expression, which affected the outcome of radiation exposure. Moreover, we developed a classifier based on two miRNAs (miR-30a and miR-126) that can reproducibly predict radiation-induced mortality. Together, we have obtained a five-miRNA composite signature that can identify irradiated macaques and predict their probability of survival.

Platinum and PARP Inhibitor Resistance Due to Overexpression of MicroRNA-622 in BRCA1-Mutant Ovarian Cancer.

High-grade serous ovarian carcinomas (HGSOCs) with BRCA1/2 mutations exhibit improved outcome and sensitivity to double-strand DNA break (DSB)-inducing agents (i.e., platinum and poly(ADP-ribose) polymerase inhibitors [PARPis]) due to an underlying defect in homologous recombination (HR). However, resistance to platinum and PARPis represents a significant barrier to the long-term survival of these patients. Although BRCA1/2-reversion mutations are a clinically validated resistance mechanism, they account for less than half of platinum-resistant BRCA1/2-mutated HGSOCs. We uncover a resistance mechanism by which a microRNA, miR-622, induces resistance to PARPis and platinum in BRCA1 mutant HGSOCs by targeting the Ku complex and restoring HR-mediated DSB repair. Physiologically, miR-622 inversely correlates with Ku expression during the cell cycle, suppressing non-homologous end-joining and facilitating HR-mediated DSB repair in S phase. Importantly, high expression of miR-622 in BRCA1-deficient HGSOCs is associated with worse outcome after platinum chemotherapy, indicating microRNA-mediated resistance through HR rescue.