The RALF2-FERONIA-MYB63 module orchestrates growth and defense in tomato roots.

Plant secreted peptides RAPID ALKALINISATION FACTORs (RALFs), which act through the receptor FERONIA (FER), play important roles in plant growth. However, it remains unclear whether and how RALF-FER contributes to the trade-off of plant growth-defense. Here, we used a variety of techniques such as CRISPR/Cas9, protein-protein interaction and transcriptional regulation methods to investigate the role of RALF2 and its receptor FER in regulating lignin deposition, root growth, and defense against Fusarium oxysporum f. sp. lycopersici (Fol) in tomato (Solanum lycopersicum). The ralf2 and fer mutants show reduced primary root length, elevated lignin accumulation, and enhanced resistance against Fol than the wild-type. FER interacts with and phosphorylates MYB63 to promote its degradation. MYB63 serves as an activator of lignin deposition by regulating the transcription of dirigent protein gene DIR19. Mutation of DIR19 suppresses lignin accumulation, and reverses the short root phenotype and Fol resistance in ralf2 or fer mutant. Collectively, our results demonstrate that the RALF2-FER-MYB63 module fine-tunes root growth and resistance against Fol through regulating the deposition of lignin in tomato roots. The study sheds new light on how plants maintain the growth-defense balance via RALF-FER.

A transcriptional regulation of ERF15 contributes to ABA-mediated cold tolerance in tomato.

Cold stress is a major meteorological threat to crop growth and yield. Abscisic acid (ABA) plays important roles in plant cold tolerance by activating the expression of cold-responsive genes; however, the underlying transcriptional regulatory module remains unknown. Here, we demonstrated that the cold- and ABA-responsive transcription factor ETHYLENE RESPONSE FACTOR 15 (ERF15) positively regulates ABA-mediated cold tolerance in tomato. Exogenous ABA treatment significantly enhanced cold tolerance in wild-type tomato plants but failed to rescue erf15 mutants from cold stress. Transcriptome analysis showed that ERF15 was associated with the expression of cold-responsive transcription factors such as CBF1 and WRKY6. Further RT-qPCR assays confirmed that the ABA-induced increased in CBF1 and WRKY6 transcripts was suppressed in erf15 mutants when the plants were subjected to cold treatment. Moreover, yeast one-hybrid assays, dual-luciferase assays and electrophoretic mobility shift assays demonstrated that ERF15 activated the transcription of CBF1 and WRKY6 by binding their promoters. Silencing CBF1 or WRKY6 significantly decreased cold tolerance. Overall, our study identified the role of ERF15 in conferring ABA-mediated cold tolerance in tomato plants by activating CBF1 and WRKY6 expression. This study not only broadens our knowledge of the mechanism of ABA-mediated cold tolerance in plants but also highlights ERF15 as an ideal target gene for cold-tolerant crop breeding.

Conserved and divergent evolution of the bZIP transcription factor in five diploid Gossypium species.

MAIN CONCLUSION: 495 bZIP members with 12 subfamilies were identified in the five diploid cottons. Segmental duplication events in cotton ancestor might have led to primary expansion of the cotton bZIP members. The basic leucine zipper (bZIP) transcription factor is one of the largest and most diverse families in plants. The evolutionary history of the bZIP family is still unclear in cotton. In this study, a total of 495 bZIP members were identified in five diploid Gossypium species, including 100 members in Gossypium arboreum, 104 members in Gossypium herbaceum, 95 members in Gossypium raimondii, 96 members in Gossypium longicalyx, and 100 members in Gossypium turneri. The bZIP members could be divided into 12 subfamilies with biased gene proportions, gene structures, conserved motifs, expansion rates, gene loss rates, and cis-regulatory elements. A total of 239 duplication events were identified in the five Gossypium species, and mainly occurred in their common ancestor. Furthermore, some GabZIPs and GhebZIPs could be regarded as important candidates in cotton breeding. The bZIP members had a conserved and divergent evolution in the five diploid Gossypium species. The current study laid an important foundation on the evolutionary history of the bZIP family in cotton.

Herbivore-induced Ca2+ signals trigger a jasmonate burst by activating ERF16-mediated expression in tomato.

Herbivory severely affects plant growth, posing a threat to crop production. Calcium ion (Ca2+ ) signaling and accumulation of jasmonates (JAs) are activated in plant response to herbivore attack, leading to the expression of defense pathways. However, little is known about how the Ca2+ signal modulates JA biosynthesis. We used diverse techniques, including CRISPR/Cas9, UPLC-MS/MS and molecular biology methods to explore the role of ETHYLENE RESPONSE FACTOR 16 in Ca2+ signal-triggered JA burst during herbivore defense in tomato. Here we show that simulated herbivory induces GLUTAMATE RECEPTOR LIKE3.3/3.5 (GLR3.3/3.5)-dependent increases in electrical activity, Ca2+ influx and increases the abundance of CALMODULIN2 (CaM2) and ERF16 transcripts in tomato. The interaction between CaM2 and ERF16 promotes JA biosynthesis by enhancing the transcriptional activity of ERF16, which increases the activation of ERF16 expression and causes expression of LIPOXYGENASE D (LOXD), AOC and 12-OXO-PHYTODIENOIC ACID REDUCTASE 3 (OPR3), the key genes in JA biosynthesis. Mutation of CaM2 results in decreased JA accumulation, together with the expression of JA biosynthesis-related genes, leading to reduced resistance to the cotton bollworm Helicoverpa armigera. These findings reveal a molecular mechanism underpinning the Ca2+ signal-initiated systemic JA burst and emphasize the pivotal role of Ca2+ signal/ERF16 crosstalk in herbivore defense.

Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding.

The MDMX oncoprotein is an important regulator of tumor suppressor p53 activity during embryonic development. Despite sequence homology to the ubiquitin E3 ligase MDM2, MDMX depletion activates p53 without significant increase in p53 level, implicating a degradation-independent mechanism. We present evidence that MDMX inhibits the sequence-specific DNA binding activity of p53. This function requires the cooperation between MDMX and CK1α, and phosphorylation of S289 on MDMX. Depletion of MDMX or CK1α increases p53 DNA binding without stabilization of p53. A proteolytic fragment release assay revealed that in the MDMX-p53 complex, the MDMX acidic domain and RING domain interact stably with the p53 DNA binding domain. These interactions are referred to as secondary interactions because they only occur after the canonical-specific binding between the MDMX and p53 N termini, but exhibit significant binding stability in the mature complex. CK1α cooperates with MDMX to inhibit p53 DNA binding by further stabilizing the MDMX acidic domain and p53 core domain interaction. These results suggest that secondary intermolecular interaction is important in p53 regulation by MDMX, which may represent a common phenomenon in complexes containing multidomain proteins.

Dynamic and epistatic QTL mapping reveals the complex genetic architecture of waterlogging tolerance in chrysanthemum.

MAIN CONCLUSION: 37 unconditional QTLs, 51 conditional QTLs and considerable epistatic QTLs were detected for waterlogging tolerance, and six favourable combinations were selected accelerating the possible application of MAS in chrysanthemum breeding. Chrysanthemum is seriously impacted by soil waterlogging. To determine the genetic characteristics of waterlogging tolerance (WAT) in chrysanthemum, a population of 162 F1 lines was used to construct a genetic map to identify the dynamic and epistatic quantitative trait loci (QTLs) for four WAT traits: wilting index (WI), dead leaf ratio (DLR), chlorosis score (Score) and membership function value of waterlogging (MFVW). The h B2 for the WAT traits ranged from 0.49 to 0.64, and transgressive segregation was observed in both directions. A total of 37 unconditional consensus QTLs with 5.81-18.21% phenotypic variation explanation (PVE) and 51 conditional consensus QTLs with 5.90-24.56% PVE were detected. Interestingly, three unconditional consensus QTLs were consistently identified across different stages, whereas no conditional consensus QTLs were consistently expressed. In addition, considerable epistatic QTLs, all with PVE values ranging from 0.01 to 8.87%, were detected by a joint analysis of WAT phenotypes. These results illustrated that the QTLs (genes) controlling WAT were environmentally dependent and selectively expressed at different times and indicated that both additive and epistatic effects underlie the inheritance of WAT in chrysanthemum. The findings of the current study provide insights into the complex genetic architecture of WAT, and the identification of favourable alleles represents an important step towards the application of molecular marker-assisted selection (MAS) and QTL pyramiding in chrysanthemum WAT breeding programmes.

Autoinhibition of MDMX by intramolecular p53 mimicry.

The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.

Preventive and therapeutic effects of adenanthin on experimental autoimmune encephalomyelitis by inhibiting NF-κB signaling.

Adenanthin, a diterpenoid isolated from the leaves of Isodon adenanthus, has been reported to possess antileukemic activity through targeting peroxiredoxin I/II. However, its other potential activities remain to be explored. Using myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we report in this study that adenanthin exerts efficaciously preventive and therapeutic effects on EAE accompanied by significant restriction of infiltration of inflammatory cells and demyelination in CNS. Adenanthin-presented immunomodulatory effects on EAE are correlated with suppressed proliferation of MOG35-55-reactive T cells, decreased Th1 and Th17 cells, increased regulatory T cell populations, decreased production of serum proinflammatory cytokines, and reduced stimulatory capacity of APCs, which might be mediated by its inhibitory action on NF-κB signaling pathway. Our results propose that, as a novel NF-κB inhibitor, adenanthin has potent immunomodulatory activity for the treatment of multiple sclerosis and possibly other autoimmune disorders.

[Genotypes of human papilloma virus in male condyloma acuminata patients in Lishui area].

OBJECTIVE: To explore the genotypes of human papilloma virus (HPV) in male patients with condyloma acuminate in Lishui area of Zhejiang Province, China. METHODS: Using HybriMax, we identified the genotypes of HPV of the verruca samples from 110 male condyloma acuminate patients aged 16 - 65 years in Lishui area. RESULTS: HPV infection was detected in 107 (97.27%) of the condyloma acuminate patients, including 76 cases (71.02%) of simple infection and 31 cases (28.97%) of multiple infection. The peak age of infection was 21 - 40 years old. Totally, 14 genotypes of HPV were identified, including the high-risk genotypes as HPV16, 53, 52, 33, CP8304, 58, 51, 45, 66, 68, and 59, which accounted for 44.85% of the cases, and the low-risk ones as HPV6, 11, and 19 (43), with an infection rate of 93.45%. CONCLUSION: The low-risk genotype is a dominant type of HPV infection and the rates of high-risk genotype and multiple infection are high among the male condyloma acuminate patients in Lishui area.

The MYC2-PUB22-JAZ4 module plays a crucial role in jasmonate signaling in tomato.

Jasmonates (JAs), a class of lipid-derived stress hormones, play a crucial role across an array of plant physiological processes and stress responses. Although JA signaling is thought to rely predominantly on the degradation of specific JAZ proteins by SCFCOI1, it remains unclear whether other pathways are involved in the regulation of JAZ protein stability. Here, we report that PUB22, a plant U-box type E3 ubiquitin ligase, plays a critical role in the regulation of plant resistance against Helicoverpa armigera and other JA responses in tomato. Whereas COI1 physically interacts with JAZ1/2/5/7, PUB22 physically interacts with JAZ1/3/4/6. PUB22 ubiquitinates JAZ4 to promote its degradation via the 26S proteasome pathway. Importantly, we observed that pub22 mutants showreduced resistance to H. armigera, whereas jaz4 single mutants and jaz1 jaz3 jaz4 jaz6 quadruple mutants have enhanced resistance. The hypersensitivity of pub22 mutants to herbivores could be partially rescued by JAZ4 mutation. Moreover, we found that expression of PUB22 can be transcriptionally activated by MYC2, thus forming a positive feedback circuit in JA signaling. We noticed that the PUB22-JAZ4 module also regulates other JA responses, including defense against B. cinerea, inhibition of root elongation, and anthocyanin accumulation. Taken together, these results indicate that PUB22 plays a crucial role in plant growth and defense responses, together with COI1-regulated JA signaling, by targeting specific JAZs.

MGMT function determines the differential response of ATR inhibitors with DNA-damaging agents in glioma stem cells for GBM therapy.

Background: The most prevalent cancer treatments cause cell death through DNA damage. However, DNA damage response (DDR) repair pathways, initiated by tumor cells, can withstand the effects of anticancer drugs, providing justification for combining DDR inhibitors with DNA-damaging anticancer treatments. Methods: Cell viability assays were performed with CellTiter-Glo assay. DNA damage was evaluated using Western blotting analysis. RNA-seq and single-cell level expression were used to identify the DDR signatures. In vivo, studies were conducted in mice to determine the effect of ATris on TMZ sensitization. Results: We found a subpopulation of glioma sphere-forming cells (GSCs) with substantial synergism with temozolomide (TMZ) using a panel of 3 clinical-grade ataxia-telangiectasia- and Rad3-related kinase inhibitors (ATRis), (elimusertib, berzosertib, and ceralasertib). Interestingly, most synergistic cell lines had O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, indicating that ATRi mainly benefits tumors with no MGMT repair. Further, TMZ activated the ATR-checkpoint kinase 1 (Chk1) axis in an MGMT-dependent way. TMZ caused ATR-dependent Chk1 phosphorylation and DNA double-strand breaks as shown by increased γH2AX. Increased DNA damage and decreased Chk1 phosphorylation were observed upon the addition of ATRis to TMZ in MGMT-methylated (MGMT-) GSCs. TMZ also improved sensitivity to ATRis in vivo, as shown by increased mouse survival with the TMZ and ATRi combination treatment. Conclusions: This research provides a rationale for selectively targeting MGMT-methylated cells using ATRis and TMZ combination. Overall, we believe that MGMT methylation status in GBM could serve as a robust biomarker for patient selection for ATRi combined with TMZ.

Circular RNA hsa_circ_0000144 aggravates ovarian Cancer progression by regulating ELK3 via sponging miR-610.

BACKGROUND: Ovarian cancer is a common cause of death among women and a health problem worldwide. Circ_0000144 has been confirmed to be an oncogene involved in cancer progression, such as gastric cancer. However, the role of circ_0000144 in ovarian cancer remains unclear and needs to be elucidated. This retrospective study aimed to investigate the underlying mechanism of circ_0000144 in ovarian cancer. METHODS: Differentially expressed circ_0000144 expression in ovarian cancer and normal tissues was identified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). In vitro assays were performed to explore the biological functions of circ_0000144 in ovarian cancer cells. An in vivo xenograft model was used to investigate the efficacy of circ_0000144 in the progression of ovarian cancer. RESULTS: Circ_0000144 was significantly upregulated in ovarian cancer cells and tissues. Circ_0000144 overexpression significantly promoted ovarian cancer cell proliferation, migration, and invasion. This study further demonstrated that circ_0000144 downregulated ELK3 levels by sponging miR-610 in ovarian cancer cells. Moreover, circ_0000144 significantly promotes ovarian cancer tumorigenesis in vivo. CONCLUSION: Our data indicate that circ_0000144 could enhance the carcinogenesis of ovarian cancer by specifically targeting miR-610, which may serve as a novel target for the diagnosis and prognosis of ovarian cancer.

Rosmarinic Acid Delays Tomato Fruit Ripening by Regulating Ripening-Associated Traits.

Fruits are excellent sources of essential vitamins and health-boosting minerals. Recently, regulation of fruit ripening by both internal and external cues for the improvement of fruit quality and shelf life has received considerable attention. Rosmarinic acid (RA) is a kind of natural plant-derived polyphenol, widely used in the drug therapy and food industry due to its distinct physiological functions. However, the role of RA in plant growth and development, especially at the postharvest period of fruits, remains largely unknown. Here, we demonstrated that postharvest RA treatment delayed the ripening in tomato fruits. Exogenous application of RA decreased ripening-associated ethylene production and inhibited the fruit color change from green to red based on the decline in lycopene accumulation. We also found that the degradation of sucrose and malic acid during ripening was significantly suppressed in RA-treated tomato fruits. The results of metabolite profiling showed that RA application promoted the accumulation of multiple amino acids in tomato fruits, such as aspartic acid, serine, tyrosine, and proline. Meanwhile, RA application also strengthened the antioxidant system by increasing both the activity of antioxidant enzymes and the contents of reduced forms of antioxidants. These findings not only unveiled a novel function of RA in fruit ripening, but also indicated an attractive strategy to manage and improve shelf life, flavor, and sensory evolution of tomato fruits.

Integrated analysis of telomerase enzymatic activity unravels an association with cancer stemness and proliferation.

Active telomerase is essential for stem cells and most cancers to maintain telomeres. The enzymatic activity of telomerase is related but not equivalent to the expression of TERT, the catalytic subunit of the complex. Here we show that telomerase enzymatic activity can be robustly estimated from the expression of a 13-gene signature. We demonstrate the validity of the expression-based approach, named EXTEND, using cell lines, cancer samples, and non-neoplastic samples. When applied to over 9,000 tumors and single cells, we find a strong correlation between telomerase activity and cancer stemness. This correlation is largely driven by a small population of proliferating cancer cells that exhibits both high telomerase activity and cancer stemness. This study establishes a computational framework for quantifying telomerase enzymatic activity and provides new insights into the relationships among telomerase, cancer proliferation, and stemness.

PARP-mediated PARylation of MGMT is critical to promote repair of temozolomide-induced O6-methylguanine DNA damage in glioblastoma.

BACKGROUND: Temozolomide (TMZ) resistance in glioblastoma multiforme (GBM) is mediated by the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation (occurs in about 40% of patients) is associated with loss of MGMT expression (MGMT-) that compromises DNA repair, leading to a favorable response to TMZ therapy. The 60% of patients with unmethylated MGMT (MGMT+) GBM experience resistance to TMZ; in these patients, understanding the mechanism of MGMT-mediated repair and modulating MGMT activity may lead to enhanced TMZ activity. Here, we report a novel mode of regulation of MGMT protein activity by poly(ADP-ribose) polymerase (PARP). METHODS: MGMT-PARP interaction was detected by co-immunoprecipitation. PARylation of MGMT and PARP was detected by co-immunoprecipitation with anti-PAR antibody. O6-methylguanine (O6-MetG) adducts were quantified by immunofluorescence assay. In vivo studies were conducted in mice to determine the effectiveness of PARP inhibition in sensitizing GBM to TMZ. RESULTS: We demonstrated that PARP physically binds with MGMT and PARylates MGMT in response to TMZ treatment. In addition, PARylation of MGMT by PARP is required for MGMT binding to chromatin to enhance the removal of O6-MetG adducts from DNA after TMZ treatment. PARP inhibitors reduced PARP-MGMT binding and MGMT PARylation, silencing MGMT activity to repair O6-MetG. PARP inhibition restored TMZ sensitivity in vivo in MGMT-expressing GBM. CONCLUSION: This study demonstrated that PARylation of MGMT by PARP is critical for repairing TMZ-induced O6-MetG, and inhibition of PARylation by PARP inhibitor reduces MGMT function rendering sensitization to TMZ, providing a rationale for combining PARP inhibitors to sensitize TMZ in MGMT-unmethylated GBM.

EGFR Amplification Induces Increased DNA Damage Response and Renders Selective Sensitivity to Talazoparib (PARP Inhibitor) in Glioblastoma.

PURPOSE: Exploration of novel strategies to extend the benefit of PARP inhibitors beyond BRCA-mutant cancers is of great interest in personalized medicine. Here, we identified EGFR amplification as a potential biomarker to predict sensitivity to PARP inhibition, providing selection for the glioblastoma (GBM) patient population who will benefit from PARP inhibition therapy. EXPERIMENTAL DESIGN: Selective sensitivity to the PARP inhibitor talazoparib was screened and validated in two sets [test set (n = 14) and validation set (n = 13)] of well-characterized patient-derived glioma sphere-forming cells (GSC). FISH was used to detect EGFR copy number. DNA damage response following talazoparib treatment was evaluated by γH2AX and 53BP1 staining and neutral comet assay. PARP-DNA trapping was analyzed by subcellular fractionation. The selective monotherapy of talazoparib was confirmed using in vivo glioma models. RESULTS: EGFR-amplified GSCs showed remarkable sensitivity to talazoparib treatment. EGFR amplification was associated with increased reactive oxygen species (ROS) and subsequent increased basal expression of DNA-repair pathways to counterelevated oxidative stress, and thus rendered vulnerability to PARP inhibition. Following talazoparib treatment, EGFR-amplified GSCs showed enhanced DNA damage and increased PARP-DNA trapping, which augmented the cytotoxicity. EGFR amplification-associated selective sensitivity was further supported by the in vivo experimental results showing that talazoparib significantly suppressed tumor growth in EGFR-amplified subcutaneous models but not in nonamplified models. CONCLUSIONS: EGFR-amplified cells are highly sensitive to talazoparib. Our data provide insight into the potential of using EGFR amplification as a selection biomarker for the development of personalized therapy.

Adaptive super-twisting sliding mode observer based robust backstepping sensorless speed control for IPMSM.

This paper proposes a higher-order sliding mode observer based robust backstepping control to realize high-performance sensorless speed regulation for the interior permanent magnet synchronous motor (IPMSM). A new robust adaptive super-twisting higher-order sliding mode based observer is proposed to estimate the rotor position. The proposed observer has advantages of sliding chattering reduction and robustness against uncertainties. And, a new robust integral adaptive backstepping control with sliding mode actions is designed to achieve precise speed regulation. The uncertainties with unknown bounds can be stabilized by the sliding mode actions. And both transient and steady performance can be achieved by using the sliding mode and integral actions simultaneously. Then, a sensorless scheme is put forward to by combining the presented observer and the proposed controller. The stability of the observer and controller are verified. Simulation and experiment results validate the proposed approach.

BRCA1 identified as a modulator of temozolomide resistance in P53 wild-type GBM using a high-throughput shRNA-based synthetic lethality screening.

Glioblastoma multiforme (GBM), the most common type of primary brain tumor, is universally fatal, with a median survival duration ranging from 12-15 months despite maximum treatment efforts. Temozolomide (TMZ) is the current standard of care for GBM patients; however patients usually develop resistance to TMZ and limits its benefit. The identification of novel synergistic targets in GBM will lead to the development of new targeted drugs, which could be combined with broad-spectrum cytotoxic agents. In this study, we used a high-throughput synthetic lethality screen with a pooled short hairpin DNA repair library, in combination with TMZ, to identify targets that will enhance TMZ-induced antitumor effects. Using an unbiased bioinformatical analysis, we identified BRCA1 as a potential promising candidate gene that induced synthetic lethality with TMZ in glioma sphere-forming cells (GSCs). BRCA1 knockdown resulted in antitumor activity with TMZ in P53 wild-type GSCs but not in P53 mutant GSCs. TMZ treatment induced a DNA damage repair response; the activation of BRCA1 DNA repair pathway targets and knockdown of BRCA1, together with TMZ, led to increased DNA damage and cell death in P53 wild-type GSCs. Our study identified BRCA1 as a potential target that sensitizes TMZ-induced cell death in P53 wild-type GBM, suggesting that the combined inhibition of BRCA1 and TMZ treatment will be a successful targeted therapy for GBM patients.

Wild-type TP53 defined gamma-secretase inhibitor sensitivity and synergistic activity with doxorubicin in GSCs.

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. Aggressive surgical resection plus radiotherapy and temozolomide have prolonged patients' median survival to only 14.6 months. Therefore, there is a critical need to develop novel therapeutic strategies for GBM. In this study, we evaluated the effect of NOTCH signaling intervention by gamma-secretase inhibitors (GSIs) on glioma sphere-forming cells (GSCs). GSI sensitivity exhibited remarkable selectivity among wild-type TP53 (wt-p53) GSCs. GSIs significantly impaired the sphere formation of GSCs harboring wt-p53. We also identified a concurrence between GSI sensitivity, NOTCH1 expression, and wt-p53 activity in GSCs. Through a series of gene editing and drug treatment experiments, we found that wt-p53 did not modulate NOTCH1 pathway, whereas NOTCH1 signaling positively regulated wt-p53 expression and activity in GSCs. Finally, GSIs (targeting NOTCH signaling) synergized with doxorubicin (activating wt-p53) to inhibit proliferation and induce apoptosis in wt-p53 GSCs. Taken together, we identified wt-p53 as a potential marker for GSI sensitivity in GSCs. Combining GSI with doxorubicin synergistically inhibited the proliferation and survival of GSCs harboring wt-p53.

Tie2-FGFR1 Interaction Induces Adaptive PI3K Inhibitor Resistance by Upregulating Aurora A/PLK1/CDK1 Signaling in Glioblastoma.

PI3K-targeting therapy represents one of the most sought-after therapies for glioblastoma (GBM). Several small-molecule inhibitors have been evaluated in clinical trials, however, the emergence of resistance limits treatment potential. Here, we generated a patient-derived glioma sphere-forming cell (GSC) xenograft model resistant to the PI3K-specific inhibitor BKM-120. Integrated RNA sequencing and high-throughput drug screening revealed that the Aurora A kinase (Aurora A)/Polo-like kinase 1 (PLK1)/cyclin-dependent kinase 1 (CDK1) signaling pathway was the main driver of PI3K inhibitor resistance in the resistant xenografts. Aurora kinase was upregulated and pCDK1 was downregulated in resistant tumors from both xenografts and tumor tissues from patients treated with the PI3K inhibitor. Mechanistically, the tyrosine kinase receptor Tie2 physically interacted with FGFR1, promoting STAT3 phosphorylation and binding to the AURKA promoter, which increased Aurora A expression in resistant GSCs. Concurrent inhibition of Aurora A and PI3K signaling overcame PI3K inhibitor-induced resistance. This study offers a proof of concept to target PI3K and the collateral-activated pathway to improve GBM therapy. SIGNIFICANCE: These findings provide novel insights into the mechanisms of PI3K inhibitor resistance in glioblastoma.