CAR-T Therapies in Solid Tumors: Opportunities and Challenges.

PURPOSE OF REVIEW: This review will discuss the challenges facing chimeric antigen receptor (CAR)-T cell application for solid tumors and opportunities to overcome these obstacles. In addition, this review will examine therapies that are in development for pediatric solid tumors. RECENT FINDINGS: The similar success of CAR-T cell treatment for hematological malignancies has not been observed in solid tumors because of the hostile tumor microenvironment and tumor heterogeneity. Most strategies developed to combat these limitations emphasize combinatorial techniques that still require further testing. Preliminary results of multiple clinical trials, including GD2- and HER2-CAR-T cells, are encouraging but must be reproduced and validated on a larger scale. CAR-T cell application in solid tumors remains challenging, and most research is in development. Several clinical trials are ongoing for pediatric solid tumors. Early results are promising but demonstrate the need for CAR-T cell modification to prevent tumor recurrence.

CAR T-cells to treat brain tumors.

Tremendous success using CAR T therapy in hematological malignancies has garnered significant interest in developing such treatments for solid tumors, including brain tumors. This success, however, has yet to be mirrored in solid organ neoplasms. CAR T function has shown limited efficacy against brain tumors due to several factors including the immunosuppressive tumor microenvironment, blood-brain barrier, and tumor-antigen heterogeneity. Despite these considerations, CAR T-cell therapy has the potential to be implemented as a treatment modality for brain tumors. Here, we review adult and pediatric brain tumors, including glioblastoma, diffuse midline gliomas, and medulloblastomas that continue to portend a grim prognosis. We describe insights gained from different preclinical models using CAR T therapy against various brain tumors and results gathered from ongoing clinical trials. Furthermore, we outline the challenges limiting CAR T therapy success against brain tumors and summarize advancements made to overcome these obstacles.

Anti-glioblastoma activity of monensin and its analogs in an organoid model of cancer.

Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3ß, increased É£H2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.

Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

A Functional Precision Medicine Pipeline Combines Comparative Transcriptomics and Tumor Organoid Modeling to Identify Bespoke Treatment Strategies for Glioblastoma.

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30-50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

Single and double modified salinomycin analogs target stem-like cells in 2D and 3D breast cancer models.

Breast cancer remains one of the leading cancers among women. Cancer stem cells (CSCs) are tumor-initiating cells which drive progression, metastasis, and reoccurrence of the disease. CSCs are resistant to conventional chemo- and radio-therapies and their ability to survive such treatment enables tumor reestablishment. Metastasis is the main cause of mortality in women with breast cancer, thus advances in treatment will depend on therapeutic strategies targeting CSCs. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic known for its anticancer activity towards several types of tumor cells. In the present work, a library of 17 C1-single and C1/C20-double modified SAL analogs was screened to identify compounds with improved activity against breast CSCs. Six single- and two double-modified analogs were more potent (IC50 range of 1.1 ± 0.1-1.4 ± 0.2 µM) toward the breast cancer cell line MDA-MB-231 compared to SAL (IC50 of 4.9 ± 1.6 µM). Double-modified compound 17 was found to be more efficacious than SAL against the majority of cancer cell lines in the NCI-60 Human Tumor Cell Line Panel. Compound 17 was more potent than SAL in inhibiting cell migration and cell renewal properties of MDA-MB-231 cells, as well as inducing selective loss of the CD44+/CD24/low stem-cell-like subpopulation in both monolayer (2D) and organoid (3D) culture. The present findings highlight the therapeutic potential of SAL analogs towards breast CSCs and identify select compounds that merit further study and clinical development.

Inhibition of tryptophan 2,3-dioxygenase impairs DNA damage tolerance and repair in glioma cells.

Expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy in gliomas through kynurenine (KYN) signaling. We report that inhibition of TDO activity attenuated recovery from replication stress and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU). Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to BCNU treatment, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced-indicative of increased fork collapse. Analysis of quantitative proteomic results revealed that TDO inhibition reduced nuclear 53BP1 and sirtuin levels. We confirmed that cells lacking TDO activity exhibited elevated gamma-H2AX signal and defective recruitment of 53BP1 to chromatin following BCNU treatment, which corresponded with delayed repair of DNA breaks. Addition of exogenous KYN increased the rate of break repair. TDO inhibition diminished SIRT7 deacetylase recruitment to chromatin, which increased histone H3K18 acetylation-a key mark involved in preventing 53BP1 recruitment to sites of DNA damage. TDO inhibition also sensitized cells to ionizing radiation (IR)-induced damage, but this effect did not involve altered 53BP1 recruitment. These experiments support a model where TDO-mediated KYN signaling helps fuel a robust response to replication stress and DNA damage.

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

Inhibition of Human DNA Polymerases Eta and Kappa by Indole-Derived Molecules Occurs through Distinct Mechanisms.

Overexpression of human DNA polymerase kappa (hpol κ) in glioblastoma is associated with shorter survival time and resistance to the alkylating agent temozolomide (TMZ), making it an attractive target for the development of small-molecule inhibitors. We previously reported on the development and characterization of indole barbituric acid-derived (IBA) inhibitors of translesion DNA synthesis polymerases (TLS pols). We have now identified a potent and selective inhibitor of hpol κ based on the indole-aminoguanidine (IAG) chemical scaffold. The most promising IAG analogue, IAG-10, exhibited greater inhibitory action against hpol κ than any other human Y-family member, as well as pols from the A-, B-, and X-families. Inhibition of hpol κ by IAG analogues appears to proceed through a mechanism that is distinct from inhibition of hpol η based on changes in DNA binding affinity and nucleotide insertion kinetics. By way of comparison, both IAG and IBA analogues inhibited binary complex formation by hpol κ and ternary complex formation by hpol η. Decreasing the concentration of enzyme and DNA in the reaction mixture lowered the IC50 value of IAG-10 to submicromolar values, consistent with inhibition of binary complex formation for hpol κ. Chemical footprinting experiments revealed that IAG-10 binds to a cleft between the finger, little finger, and N-clasp domains on hpol κ and that this likely disrupts the interaction between the N-clasp and the TLS pol core. In cell culture, IAG-10 potentiated the antiproliferative activity and DNA damaging effects of TMZ in hpol κ-proficient cells but not in hpol κ-deficient cells, indicative of a target-dependent effect. Mutagenic replication across alkylation damage increased in hpol κ-proficient cells treated with IAG-10, while no change in mutation frequency was observed for hpol κ-deficient cells. In summary, we developed a potent and selective small-molecule inhibitor of hpol κ that takes advantage of structural features unique to this TLS enzyme to potentiate TMZ, a standard-of-care drug used in the treatment of malignant brain tumors. Furthermore, the IAG scaffold represents a new chemical space for the exploration of TLS pol inhibitors, which could prove useful as a strategy for improving patient response to genotoxic drugs.

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation.

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.

A Small-Molecule Inhibitor of Human DNA Polymerase η Potentiates the Effects of Cisplatin in Tumor Cells.

Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC50 value of 8 µM and exhibited 5-10-fold specificity for hpol η over replicative pols. We conclude from kinetic analyses, chemical footprinting assays, and molecular docking that PNR-7-02 binds to a site on the little finger domain and interferes with the proper orientation of template DNA to inhibit hpol η. A synergistic increase in CDDP toxicity was observed in hpol η-proficient cells co-treated with PNR-7-02 (combination index values = 0.4-0.6). Increased γH2AX formation accompanied treatment of hpol η-proficient cells with CDDP and PNR-7-02. Importantly, PNR-7-02 did not impact the effect of CDDP on cell viability or γH2AX in hpol η-deficient cells. In summary, we observed hpol η-dependent effects on DNA damage/replication stress and sensitivity to CDDP in cells treated with PNR-7-02. The ability to employ a small-molecule inhibitor of hpol η to improve the cytotoxic effect of CDDP may aid in the development of more effective chemotherapeutic strategies.

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress.

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins.

Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles.

Kynurenine Signaling Increases DNA Polymerase Kappa Expression and Promotes Genomic Instability in Glioblastoma Cells.

Overexpression of the translesion synthesis polymerase hpol κ in glioblastomas has been linked to poor patient prognosis; however, the mechanism promoting higher expression in these tumors remains unknown. We determined that activation of the aryl hydrocarbon receptor (AhR) pathway in glioblastoma cells leads to increased hpol κ mRNA and protein levels. We blocked nuclear translocation and DNA binding by AhR in glioblastoma cells using a small-molecule and observed decreased hpol κ expression. Pharmacological inhibition of tryptophan-2,3-dioxygenase (TDO), the enzyme largely responsible for activating AhR in glioblastoma, led to a decrease in the endogenous AhR agonist kynurenine and a corresponding decrease in hpol κ protein levels. Importantly, we discovered that inhibiting TDO activity, AhR signaling, or suppressing hpol κ expression with RNA interference led to decreased chromosomal damage in glioblastoma cells. Epistasis assays further supported the idea that TDO activity, activation of AhR signaling, and the resulting overexpression of hpol κ function primarily in the same pathway to increase endogenous DNA damage. These findings indicate that upregulation of hpol κ through glioblastoma-specific TDO activity and activation of AhR signaling likely contributes to the high levels of replication stress and genomic instability observed in these tumors.