High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity.

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

Bcl6, Irf2, and Notch2 promote nonclassical monocyte development.

Ly6Clo monocytes are a myeloid subset that specializes in the surveillance of vascular endothelium. Ly6Clo monocytes have been shown to derive from Ly6Chi monocytes. NOTCH2 signaling has been implicated as a trigger for Ly6Clo monocyte development, but the basis for this effect is unclear. Here, we examined the impact of NOTCH2 signaling of myeloid progenitors on the development of Ly6Clo monocytes in vitro. NOTCH2 signaling induced by delta-like ligand 1 (DLL1) efficiently induced the transition of Ly6Chi TREML4- monocytes into Ly6Clo TREML4+ monocytes. We further identified two additional transcriptional requirements for development of Ly6Clo monocytes. Deletion of BCL6 from myeloid progenitors abrogated development of Ly6Clo monocytes. IRF2 was also required for Ly6Clo monocyte development in a cell-intrinsic manner. DLL1-induced in vitro transition into Ly6Clo TREML4+ monocytes required IRF2 but unexpectedly could occur in the absence of NUR77 or BCL6. These results imply a transcriptional hierarchy for these factors in controlling Ly6Clo monocyte development.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

Bcl6-Independent In Vivo Development of Functional Type 1 Classical Dendritic Cells Supporting Tumor Rejection.

The transcriptional repressor Bcl6 has been reported as required for development of a subset of classical dendritic cell (cDCs) called cDC1, which is responsible for cross-presentation. However, mechanisms and in vivo functional analysis have been lacking. We generated a system for conditional deletion of Bcl6 in mouse cDCs. We confirmed the reported in vitro requirement for Bcl6 in cDC1 development and the general role for Bcl6 in cDC development in competitive settings. However, deletion of Bcl6 did not abrogate the in vivo development of cDC1. Instead, Bcl6 deficiency caused only a selective reduction in CD8α expression by cDC1 without affecting XCR1 or CD24 expression. Normal cDC1 development was confirmed in Bcl6cKO mice by development of XCR1+ Zbtb46-GFP+ cDC1 by rejection of syngeneic tumors and by priming of tumor-specific CD8 T cells. In summary, Bcl6 regulates a subset of cDC1-specific markers and is required in vitro but not in vivo for cDC1 development.

Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.

Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.

Notch2-dependent DC2s mediate splenic germinal center responses.

CD4+ T follicular helper (TFH) cells support germinal center (GC) reactions promoting humoral immunity. Dendritic cell (DC) diversification into genetically distinct subsets allows for specialization in promoting responses against several types of pathogens. Whether any classical DC (cDC) subset is required for humoral immunity is unknown, however. We tested several genetic models that selectively ablate distinct DC subsets in mice for their impact on splenic GC reactions. We identified a requirement for Notch2-dependent cDC2s, but not Batf3-dependent cDC1s or Klf4-dependent cDC2s, in promoting TFH and GC B cell formation in response to sheep red blood cells and inactivated Listeria monocytogenes This effect was mediated independent of Il2ra and several Notch2-dependent genes expressed in cDC2s, including Stat4 and Havcr2 Notch2 signaling during cDC2 development also substantially reduced the efficiency of cDC2s for presentation of MHC class II-restricted antigens, limiting the strength of CD4 T cell activation. Together, these results demonstrate a nonredundant role for the Notch2-dependent cDC2 subset in supporting humoral immune responses.

The role of quiescent thymic progenitors in TAL/LMO2-induced T-ALL chemotolerance.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

Definitive Radiation Therapy for Medically Inoperable Endometrial Carcinoma.

Purpose: Upfront radiation therapy consisting of brachytherapy with or without external beam radiation therapy is considered standard of care for patients with endometrial carcinoma who are unable to undergo surgical intervention. This study evaluated the cancer-free survival (CFS), cancer-specific survival (CSS), and overall survival (OS) of patients with endometrial carcinoma managed with definitive-intent radiation therapy. Methods and Materials: This was a single-institution retrospective analysis of medically inoperable patients with biopsy-proven endometrial carcinoma managed with up-front, definitive radiation therapy at UMass Memorial Medical Center between May 2010 and October 2021. A total of 55 cases were included for analysis. Patients were stratified as having low-risk endometrial carcinoma (LREC; uterine-confined grade 1-2 endometrioid adenocarcinoma) or high-risk endometrial carcinoma (HREC; stage III/IV and/or grade 3 endometrioid carcinoma, or any stage serous or clear cell carcinoma or carcinosarcoma). The CFS, CSS, OS, and grade ≥3 toxic effects were reported for patients with LREC and HREC. Results: The median age was 66 years (range, 42-86 years), and the median follow-up was 44 months (range, 4-135 months). Twelve patients (22%) were diagnosed with HREC. Six patients (11%) were treated with high-dose-rate brachytherapy alone and 49 patients (89%) were treated with high-dose-rate brachytherapy and external beam radiation therapy. Twelve patients (22%) were treated with radiation and chemotherapy. The 2-year CFS was 82% for patients with LREC and 80% for patients with HREC (log rank P = .0654). The 2-year CSS was 100% for both LREC and HREC patients. The 2-year OS was 92% for LREC and 80% for HREC (log P = .0064). There were no acute grade ≥3 toxic effects. There were 3 late grade ≥3 toxic effects owing to endometrial bleeding and gastrointestinal adverse effects. Conclusions: For medically inoperable patients with endometrial carcinoma, up-front radiation therapy provided excellent CFS, CSS, and OS. The CSS and OS were higher in patients with LREC than in those with HREC. Toxic effects were limited in both cohorts.

ESRRB regulates glucocorticoid gene expression in mice and patients with acute lymphoblastic leukemia.

Synthetic glucocorticoids (GCs), such as dexamethasone and prednisone, remain key components of therapy for patients with lymphoid malignancies. For pediatric patients with acute lymphoblastic leukemia (ALL), response to GCs remains the most reliable prognostic indicator; failure to respond to GC correlates with poor event-free survival. To uncover GC resistance mechanisms, we performed a genome-wide, survival-based short hairpin RNA screen and identified the orphan nuclear receptor estrogen-related receptor-ß (ESRRB) as a critical transcription factor that cooperates with the GC receptor (GR) to mediate the GC gene expression signature in mouse and human ALL cells. Esrrb knockdown interfered with the expression of genes that were induced and repressed by GR and resulted in GC resistance in vitro and in vivo. Dexamethasone treatment stimulated ESRRB binding to estrogen-related receptor elements (ERREs) in canonical GC-regulated genes, and H3K27Ac Hi-chromatin immunoprecipitation revealed increased interactions between GR- and ERRE-containing regulatory regions in dexamethasone-treated human T-ALL cells. Furthermore, ESRRB agonists enhanced GC target gene expression and synergized with dexamethasone to induce leukemic cell death, indicating that ESRRB agonists may overcome GC resistance in ALL, and potentially, in other lymphoid malignancies.

TGF-ß Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia.

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by progressive bone marrow failure (BMF) from hematopoietic stem and progenitor cell (HSPC) attrition. A greater understanding of the pathogenesis of BMF could improve the therapeutic options for FA patients. Using a genome-wide shRNA screen in human FA fibroblasts, we identify transforming growth factor-ß (TGF-ß) pathway-mediated growth suppression as a cause of BMF in FA. Blocking the TGF-ß pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-ß signaling in FA HSPCs results in elevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth. Together, our results suggest that elevated TGF-ß signaling contributes to BMF in FA by impairing HSPC function and may be a potential therapeutic target for the treatment of FA.

A unique subset of epithelial ovarian cancers with platinum sensitivity and PARP inhibitor resistance.

Platinum and PARP inhibitor (PARPi) sensitivity commonly coexist in epithelial ovarian cancer (EOC) due to the high prevalence of alterations in the homologous recombination (HR) DNA repair pathway that confer sensitivity to both drugs. In this report, we describe a unique subset of EOC with alterations in another DNA repair pathway, the nucleotide excision repair (NER) pathway, which may exhibit a discordance in sensitivities to these drugs. Specifically, 8% of high-grade serous EOC from The Cancer Genome Atlas dataset exhibited NER alterations, including nonsynonymous or splice site mutations and homozygous deletions of NER genes. Tumors with NER alterations were associated with improved overall survival (OS) and progression-free survival (PFS), compared with patients without NER alterations or BRCA1/2 mutations. Furthermore, patients with tumors with NER alterations had similar OS and PFS as BRCA1/2-mutated patients, suggesting that NER pathway inactivation in EOC conferred enhanced platinum sensitivity, similar to BRCA1/2-mutated tumors. Moreover, two NER mutations (ERCC6-Q524* and ERCC4-A583T), identified in the two most platinum-sensitive tumors, were functionally associated with platinum sensitivity in vitro. Importantly, neither NER alteration affected HR or conferred sensitivity to PARPi or other double-strand break-inducing agents. Overall, our findings reveal a new mechanism of platinum sensitivity in EOC that, unlike defective HR, may lead to a discordance in sensitivity to platinum and PARPi, with potential implications for previously reported and ongoing PARPi trials in this disease.

Genetic witness: forensic uses of DNA tests.

"Genetic Witness: Forensic Uses of DNA Tests" summarizes the findings of a 204-page report by the U.S. Congressional Office of Technology Assessment (OTA). It reviews the DNA techniques used in criminal casework, evaluates the validity and reliability of the technologies, examines issues of quality assurance, reviews the legal implications of the use of DNA tests by U.S. courts, and analyzes the privacy implications of forensic DNA tests and computer databanks. It presents a range of actions that could be taken by the U.S. Congress to address five policy issues: standards for forensic uses of DNA typing; funding of crime laboratories, forensic personnel training, and forensic research; the advisability of establishing computer databanks of DNA test results; and privacy considerations of collecting, using, and storing DNA data or samples.

Somatic ERCC2 mutations correlate with cisplatin sensitivity in muscle-invasive urothelial carcinoma.

UNLABELLED: Cisplatin-based chemotherapy is the standard of care for patients with muscle-invasive urothelial carcinoma. Pathologic downstaging to pT0/pTis after neoadjuvant cisplatin-based chemotherapy is associated with improved survival, although molecular determinants of cisplatin response are incompletely understood. We performed whole-exome sequencing on pretreatment tumor and germline DNA from 50 patients with muscle-invasive urothelial carcinoma who received neoadjuvant cisplatin-based chemotherapy followed by cystectomy (25 pT0/pTis "responders," 25 pT2+ "nonresponders") to identify somatic mutations that occurred preferentially in responders. ERCC2, a nucleotide excision repair gene, was the only significantly mutated gene enriched in the cisplatin responders compared with nonresponders (q < 0.01). Expression of representative ERCC2 mutants in an ERCC2-deficient cell line failed to rescue cisplatin and UV sensitivity compared with wild-type ERCC2. The lack of normal ERCC2 function may contribute to cisplatin sensitivity in urothelial cancer, and somatic ERCC2 mutation status may inform cisplatin-containing regimen usage in muscle-invasive urothelial carcinoma. SIGNIFICANCE: Somatic ERCC2 mutations correlate with complete response to cisplatin-based chemosensitivity in muscle-invasive urothelial carcinoma, and clinically identified mutations lead to cisplatin sensitivity in vitro. Nucleotide excision repair pathway defects may drive exceptional response to conventional chemotherapy.

PARI overexpression promotes genomic instability and pancreatic tumorigenesis.

Treatment options for patients with pancreatic ductal adenocarcinoma (PDAC) remain limited. Therapeutic targets of interest include mutated molecules that predispose to pancreatic cancer such as KRAS and TP53. Here, we show that an element of the homologous recombination pathway of DNA repair, the PARP-binding protein C12orf48/PARI (PARPBP), is overexpressed specifically in pancreatic cancer cells where it is an appealing candidate for targeted therapy. PARI upregulation in pancreatic cancer cells or avian DT40 cells conferred DNA repair deficiency and genomic instability. Significantly, PARI silencing compromised cancer cell proliferation in vitro, leading to cell-cycle alterations associated with S-phase delay, perturbed DNA replication, and activation of the DNA damage response pathway in the absence of DNA damage stimuli. Conversely, PARI overexpression produced tolerance to DNA damage by promoting replication of damaged DNA. In a mouse xenograft model of pancreatic cancer, PARI silencing was sufficient to reduce pancreatic tumor growth in vivo. Taken together, our findings offered a preclinical proof-of-concept for PARI as candidate therapeutic target to treat PDAC.