Chemotherapy forms the backbone of treatment for Diffuse Large B Cell Lymphoma (DLBCL); however, approximately 20% of tumors are chemoresistant. Inhibitors of the DNA damage response (DDR) sensitize various tumor types to chemotherapy and show pre-clinical activity in lymphoma. DLBCL with replication stress and genomic instability are particularly sensitive to ATR and WEE1 inhibitors, making DDR inhibition a promising therapeutic avenue to enhance chemosensitivity. We therefore conducted an in-vitro screen to identify optimal chemotherapy-DDR inhibitor (DDRi) combinations in a panel of eight DLBCL cell lines. This screen utilized the Quadratic Phenotypic Optimization Platform (QPOP), an experimental-analytic method designed to identify potent drug combinations. Six DDRis targeting various pathways (ATR, ATM, CHK1/2, DNA-PK, WEE1, PARP) were screened along with six routinely used chemotherapy agents. The ATR inhibitor AZD6738 and replication stress-inducing chemotherapeutic gemcitabine (A+G) emerged as the most effective combination, inducing synergistic cell death via apoptosis across fourteen DLBCL cell lines, including gemcitabine-resistant lines. This efficacy was further demonstrated in vivo, with the A+G combination significantly reducing tumor growth in NSG mouse xenograft models. Contrary to the expected mechanism of the A+G combination causing mitotic catastrophe, flow cytometry revealed that only a small proportion of cells entered mitosis. RNA sequencing of A+G treated cell lines revealed expected suppression of cell-cycle and DNA replication-related pathways. Interestingly, the combination also strongly reversed a poor-prognostic gene expression signature characteristic of dark zone (DZ) biology in the DZ-like and gemcitabine-resistant cell lines HT and SUDHL4. This was accompanied by changes in a variety of chromatin regulation pathways. To further investigate the mechanism underlying this transcriptional shift, we performed chromatin immunoprecipitation sequencing (ChIP-seq) for the enhancer marker H3K27ac. This revealed that A+G treated cells showed a significant loss of super-enhancer marks for BCL6, a master regulatory transcription repressor in DLBCL. BCL6 is a proto-oncogene that represses genes involved in cell cycle arrest and apoptosis, allowing lymphoma cells to survive and proliferate, and its expression was significantly downregulated at the RNA level in A+G treated cells. Additionally, mass-spectrometry cellular thermal shift assays (MS-CETSA) demonstrated that gemcitabine initiated an early destabilization of BCL6 protein, which was sustained by ATR inhibition. However, only the A+G combination led to sustained loss of BCL6 protein levels, explaining improved cell kill with the reversal of the DZ signature. We hypothesize that the replication stress induced by gemcitabine treatment leads to activation of pathways that cause degradation of BCL6, which is then reinforced by transcriptional changes induced by ATR inhibition. In conclusion, we have identified a chemotherapy-DDRi combination, ATR inhibition and gemcitabine, that is highly effective in killing chemoresistant DLBCL cells in vitro and in vivo. The mechanism of synergy involves suppression of a BCL6-regulated transcriptional program driving dark zone biology. Gemcitabine is a routinely used chemotherapeutic for second-line treatment of DLBCL, and neither it nor AZD6738 shows lymphodepletion in humans. As DLBCL with DZ-like signatures show poor outcomes on standard chemo-immunotherapy, our finding that ATR inhibition with gemcitabine reverses DZ signatures suggests the possibility of this being a non-lymphodepleting genotoxic backbone for combinations with T-cell engaging bispecific antibodies.

AbstractBackgroundThe identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4.MethodsIn vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4’s interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations.ResultsPARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4’s tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss.ConclusionsPARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex—unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.

Background: Tumor-initiating cells (TIC) often elude conventional cancer treatment, which results in metastasis and cancer relapse.Recently, studies have begun to focus on the TIC population in tumors to provide better therapeutic options.Previously, we have reported the successful development of a TIC-specific probe TiY with the binding target as vimentin.While a low concentration of TiY showed a TIC visualization, at a high concentration, TiY induced selective toxicity onto TIC in vitro.In this study, we aim to assess TiY's applicability in theranostics purposes, from in vivo visualization to therapeutic effect toward TIC, in cancer mouse models.Methods: We performed cell experiments with the TIC line model derived from resected primary non-small cell lung cancer (NSCLC) patient tumor.The animal model studies were conducted in mice of NSCLC patient-derived xenograft (PDX).TiY was intravenously delivered into the mice models at different concentrations to assess its in vivo TIC-selective staining and therapeutic effect.Results: We demonstrated the TIC-selective identification and therapeutic effect of TiY in animal models.TiY treatment induced a significant ablation of the TIC population in the tumor, and further molecular study elucidated that the mechanism of TiY is through vimentin dynamics in TIC. Conclusion:The results underscore the applicability of TiY for cancer treatment by selectively targeting soluble vimentin in TIC.

The relatively quiet mutational landscape of rhabdomyosarcoma (RMS) suggests that epigenetic deregulation could be central to oncogenesis and tumour aggressiveness. Histone variants have long been recognised as important epigenetic regulators of gene expression. However, the role of histone variants in RMS has not been studied hitherto. In this study, we show that histone variant H3.3 is overexpressed in alveolar RMS (ARMS), an aggressive subtype of RMS. Functionally, knockdown of H3F3A, which encodes for H3.3, significantly impairs the ability of ARMS cells to undertake migration and invasion and reduces Rho activation. In addition, a striking reduction in metastatic tumour burden and improved survival is apparent in vivo. Using RNA-sequencing and ChIP-sequencing analyses, we identified melanoma cell adhesion molecule (MCAM/CD146) as a direct downstream target of H3.3. Loss of H3.3 resulted in a reduction in the presence of active marks and an increase in the occupancy of H1 at the MCAM promoter. Cell migration and invasion were rescued in H3F3A-depleted cells through MCAM overexpression. Moreover, we identified G9a, a lysine methyltransferase encoded by EHMT2, as an upstream regulator of H3F3A. Therefore, this study identifies a novel H3.3 dependent axis involved in ARMS metastasis. These findings establish the potential of MCAM as a therapeutic target for high-risk ARMS patients. © 2022 The Pathological Society of Great Britain and Ireland.

Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles. In multiple tissues, EGFR is stabilized by TAD and DBD mutants in the cytosolic and nuclear compartments respectively. TAD mutants promote EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol. Conversely, DBD mutants maintain EGFR activity in the nucleus, by blocking EGFR interaction with the phosphatase SHP1, triggering c-Myc and Cyclin D1 upregulation. Our findings suggest that p53 mutants carrying gain-of-function, mis-sense mutations affecting two different domains form new protein complexes that promote carcinogenesis by enhancing EGFR signaling via distinctive mechanisms, exposing clinically relevant therapeutic vulnerabilities.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

In the version of this article originally published, there is an error in Fig. 5a. Originally, ‘MAT2A’ appeared between ‘Methionine’ and ‘Homocysteine’. ‘MAT2A’ should have been ‘MTR’. The error has been corrected in the PDF and HTML versions of this article.

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC-specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial-to-mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti-TIC activity with low toxicity to non-TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.

How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-beta, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% +/- 7.35% TBX3(+) HNF4A(+) human liver bud progenitors and 81.5% +/- 3.2% FAH(+) hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah(-/-)Rag2(-/-)II2rg(-/-) mouse model of liver failure.

Self-renewing tumor-initiating cells (TICs) are thought to be responsible for tumor recurrence and chemo-resistance. Glycine decarboxylase, encoded by the GLDC gene, is reported to be overexpressed in TIC-enriched primary non-small-cell lung carcinoma (NSCLC). GLDC is a component of the mitochondrial glycine cleavage system, and its high expression is required for growth and tumorigenic capacity. Currently, there are no therapeutic agents against GLDC. As a therapeutic strategy, we have designed and tested splicing-modulating steric hindrance antisense oligonucleotides (shAONs) that efficiently induce exon skipping (half maximal inhibitory concentration [IC50] at 3.5-7 nM), disrupt the open reading frame (ORF) of GLDC transcript (predisposing it for nonsense-mediated decay), halt cell proliferation, and prevent colony formation in both A549 cells and TIC-enriched NSCLC tumor sphere cells (TS32). One candidate shAON causes 60% inhibition of tumor growth in mice transplanted with TS32. Thus, our shAONs candidates can effectively inhibit the expression of NSCLC-associated metabolic enzyme GLDC and may have promising therapeutic implications.

Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0±3.1% AFP + human liver bud progenitors and 87.3±9.4% ALBUMIN + hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5±3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in the Fah -/- Rag2 -/- Il2rg -/- mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.

Genome editing using programmable DNA endonucleases enables the engineering of eukaryotic cells and living organisms with desirable properties or traits. Among the various molecular scissors that have been developed to date, the most versatile and easy-to-use family of nucleases derives from CRISPR-Cas, which exists naturally as an adaptive immune system in bacteria. Recent advances in the CRISPR-Cas technology have expanded our ability to manipulate complex genomes for myriad biomedical and biotechnological applications. Some of these applications are time-sensitive or demand high spatial precision. Here, we describe the use of an inducible CRISPR-Cas9 system, termed iCas, which we have developed to enable rapid and tight control of genome editing in mammalian cells. The iCas system can be switched on or off as desired through the introduction or removal of the small molecule tamoxifen or its related analogs such as 4-hydroxytamoxifen (4-HT).